Specific binding agents for KSHV vIL-6 that neutralize a biological activity

ABSTRACT

A specific binding agent is provided, wherein the specific binding agent specifically binds Kaposi&#39;s sarcoma-associated herpesvirus (KSHV) interleukin-6 (vIL-6), and the specific binding agent neutralizes an activity of vIL-6. In one embodiment, the specific binding agent is an antibody. Methods are provided for using a specific binding agent that binds vIL-6, and neutralizes a biological activity of vIL-6. Methods of treatment for a KSHV-associated disorder are also provided. Methods for diagnosing a KSHV-associated disorder are provided, as are kits that include a specific binding agent of the invention. A method is also provided for testing an agent for effectiveness in treating a KSHV-associated disorder. The method includes incubating the agent with a cell free system comprising a vIL-6 receptor component and vIL-6, and comparing the binding of vIL-6 and the receptor component in the presence of the agent to binding of vIL-6 to the receptor component in the absence of the agent. A decrease in the binding of vIL-6 to the receptor component in the presence of the agent indicates that the agent is effective for treating the KSHV-associated disorder.

PRIORITY CLAIM

This is a divisional of U.S. patent application Ser. No. 10/333,121filed Jan. 14, 2003, now U.S. Pat. No. 6,939,547, which is a § 371 U.S.national stage of PCT/US01/24179 filed Jul. 31, 2001, which waspublished in English under PCT Article 21(2), which in turn claims thebenefit of U.S. Provisional Application No. 60/221,719 filed Jul. 31,2000. U.S. patent application Ser. No. 10/333,121 is incorporated byreference herein.

FIELD OF THE INVENTION

This invention relates to the field of herpesviruses, more specificallyto human herpesvirus 8 (HHV-8), also known as Kaposi's sarcomaassociated herpesvirus (KSHV), and to agents that bind the viral IL-6encoded by this virus.

BACKGROUND

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is a newlydescribed oncogenic herpesvirus originally identified in acquiredimmunodeficiency syndrome (AIDS)-associated Kaposi's sarcoma (KS)lesions (Chang et al., Science 266:1865, 1994). KSHV sequences areregularly detected in KS lesions from human immunodeficiency virus(HIV)-infected and non-infected individuals, primary effusion lymphoma(PEL), and a proportion of cases of Castleman's disease (Neipel et al.,J Virol 71:4187, 1997; Schulz, J Gen Virol 79:1573, 1998). KSHV encodesvarious proteins that have features suggesting their role in promotingcellular growth and transformation, including viral homologues of cyclinD, G-protein coupled receptor, interferon regulatory factor, macrophageinflammatory proteins and IL-6. All these viral proteins displaystructural similarities to their cellular counterparts. KSHV viral IL-6(vIL-6), encoded at open reading frame K2, has 24.8% amino acid sequenceidentity (49.7% similarity) to human IL-6 (hIL-6) and 24.2% identity(47.9% similarity) to murine IL-6 (mIL-6) (Moore et al., Science274:1739, 1996; Neipel, et al., J Virol 71:839, 1997; Nicholas et al.,Nat Med 3:287, 1997).

Cellular IL-6 acts on a wide variety of cell types, serving as a growthfactor for myeloma, plasmacytoma and B cells, and promoting the terminaldifferentiation of B cells into Ig-secreting cells (Kishimoto et al.,Blood 86:1243, 1995; Peters, et al., Blood 92:3495, 1998). This cytokinehas been implicated in the pathogenesis of several diseases, includingmultiple myeloma and rheumatoid arthritis as well as KSHV-relateddiseases (Neipel et al., J Virol 71:4187, 1997). The IL-6 family ofcytokines exerts its activities via receptor complexes that contain atleast one subunit of the signal transducing protein gp130. The membersof this family, which include IL-6 LIF, IL-11, oncostatin M (OSM),ciliary neurotrophic factor and cardiotrophin-1, are structurallyrelated and exert many overlapping biological activities (Kishimoto etal., Blood 86:1243, 1995; Peters et al., Blood 92:3495, 1998). Cellstimulation by any member of the IL-6 family of cytokines triggers homo-or hetero-dimerization of gp130. The dimerization of gp130 leads toactivation of associated cytoplasmic tyrosine kinases and subsequentmodification of transcription factors (Taga et al., Annu Rev Immunol15:797, 1997). In addition to gp30, the high affinity, signalingreceptor complexes for the IL-6-type cytokines contain at least oneother receptor subunit. IL-6 utilizes a specific α-subunit (IL-6Rα), andthe high affinity receptor-ligand complex consists of two molecules ofeach gp130, IL-6 and IL-6Rα (Hammacher et al., J. Biol Chem 273:22701,1998). The formation of such hexameric receptor-complexes occurs in allsituations in which the ligand requires a nonsignaling receptor for itsassociation with gp130.

In spite of its limited sequence homology, vIL-6 displays manybiological functions of cellular IL-6 (Aoki et al., Blood 93:4034,1999). Studies in vitro and in vivo have shown that vIL-6 can stimulatethe growth of KSHV-infected PEL cells (Jones et al., Blood 94:2871,1999), promote hematopoiesis, act as an angiogenic factor by inducingvascular endothelial growth factor (Aoki et al., Blood 93:4034, 1999),and activate STAT1, STAT3 and JAK1 phosphorylation (Molden et al., JBiol Chem 272:19625, 1997). The interactions of vIL-6 with the IL-6receptor chains gp130/IL-6Rα have been studied both in human and murinecell culture systems (Nicholas et al., Nat Med 3:287, 1997; Molden, JBiol Chem 272:19625, 1997; Burger et al., Blood 91:1858, 1998; Wan etal., J Virol 73:8268, 1999; Gage et al., AIDS 13:1851, 1999), but vIL-6directed molecules that selectively interfere with this interaction haveyet to be developed.

SUMMARY OF THE DISCLOSURE

A specific binding agent is provided, wherein the specific binding agentspecifically binds Kaposi's sarcoma-associated herpesvirus (KSHV)interleukin-6 (vIL-6), and the specific binding agent neutralizes anactivity of vIL-6. In one embodiment, the specific binding agent is anantibody.

Methods are provided for using a specific binding agent that bindsvIL-6, and neutralizes a biological activity of vIL-6. For example,methods for diagnosing a KSHV-associated disorder are provided, as arekits that include a specific binding agent of the invention.

In another embodiment, a method is provided for testing an agent foreffectiveness in treating a KSHV-associated disorder. The methodincludes incubating the agent with a cell free system comprising vIL-6and gp130, and comparing the binding of vIL-6 and gp130 in the presenceof the agent to binding of vIL-6 to gp130 in the absence of the agent. Adecrease in the binding of vIL-6 to gp130 in the presence of the agentindicates that the agent is effective for treating the KSHV-associateddisorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph demonstrating the binding of hIL-6 and vIL-6 tosIL-6R chains as detected by ELISA. Purified sIL-6Rα (5 μg/ml) andsgp130 (5 μg/ml) were immobilized on an ELISA plate and incubated withhIL-6 (50 ng/ml) or MBPvIL-6 (50 ng/ml). Bound protein was detected byrabbit anti-human or anti-viral IL-6 antibodies, followed byHRP-conjugated anti-rabbit IgG Abs. Panel A shows the results usingbound protein detected with a rabbit polyclonal anti-hIL-6 antibody thatrecognizes hIL-6 but not vIL-6. Panel B shows the results using apolyclonal anti-MBPvIL-6 Ab which recognizes vIL-6 but not hIL-6. Theresults represent the means of triplicate assays; error bars representSD. Representative data of three independent experiments are shown.

FIG. 2 is a graph of the neutralizing activity of mAbs against vIL-6 inthe B9 cell bioassay. Exponentially growing B9 cells (2×10³ cells/well)were cultured in medium supplemented with MBPvIL-6 (100 ng/ml) with orwithout monoclonal anti-vIL-6 Ab (0.8 to 10 μg/ml). [³H]-thymidine wasadded during the final 6 hours of culture. The results represent themean radioactivity of triplicate cultures; SDs were within 5% of themean. Representative data of two independent experiments are shown.

FIG. 3 is a series of bar graphs demonstrating the effect of mAbsagainst vIL-6 on IgM secretion by SKW6.4 cells. FIG. 3A displays theresults obtained when SKW6.4 cells (1×10⁴ cells/well) were incubated inthe presence of hIL-6 or MBPvIL-6 at various concentrations, and humanIgM production in the supernatants was measured by human IgM ELISA. FIG.3B displays the results obtained when SKW6.4 cells were incubated in thepresence of MBPvIL-6 (2 μg/ml) and mAbs against vIL-6 (0 to 10 μg/ml).The results represent the means of triplicate cultures; error barsrepresent SDs. The asterisk (*) denotes the occurrence of a significantdecrease in IgM secretion in cultures containing anti-vIL-6 mAbs(p<0.0005), compared to cultures containing MBPvIL-6 (2 μg/ml) alone.

FIG. 4 is a schematic representation of deletion mutants of vIL-6 fusionproteins. Numbers above each box represent the amino acid positionsrelative to the start methionine. Restriction enzyme sites Aat II andEcoRI used to construct M3 and M5, respectively, are indicated. Thereactivity of each monoclonal antibody (mAb) to each fusion protein wasdetermined by ELISA. The reactivity of each nab to each fusion proteinis shown as positive (+) when the absorbance (A_(405/550)) wassignificantly higher (0.7 absorbance units) than the background ornegative (−) when the absorbance was similar (within 0.05 absorbanceunits) to the background. The black box represents the putative vIL-6signal peptide portion (Neipel et al., J Virol. 71:839, 1997). Thedotted box denotes 13 amino acids of either a hIL-6 (SEQ ID NO: 1) or avIL-6 fragment (SEQ ID NO:3) that is recognized by 4 neutralizing mAbsagainst vIL-6. The helix B sequence for hIL-6 (SEQ ID NO:2) and vIL-6(SEQ ID NO:4) is shown.

FIG. 5 is a bar graph demonstrating the interference of vIL-6 binding tosgp130 by neutralizing mAbs against vIL-6. Biotinylated MBPvIL-6 (50ng/ml) was first incubated with anti-vIL-6 mAbs (20 μg/ml) or isotypecontrol mouse IgG1 (20 μg/ml), and then added onto ELISA wells coatedwith purified sgp130(2 μg/ml) in triplicates. Bound protein was detectedby streptavidin-HRP. The results represent the means±SDs. The asterisk(*) indicates the occurrence of a significant decrease in OD_(450/630)(p<0.002), compared to control Ab. Representative data of twoindependent experiments are shown.

FIG. 6 is a set of two graphs demonstrating antibody specificity forvIL-6 and establishment of a vIL-6 ELISA. FIG. 6 A shows the detectionof vIL-6 by a solid-phase sandwich ELISA using an anti-vIL-6 mousemonoclonal and a rabbit polyclonal antibodies. The lower limit ofsensitivity (the minimum amount of protein detected with 95% confidence)was calculated at 43.8 pg/mL MBP-vIL-6, corresponding to approximately14.7 pg/ml of vIL-6. The assay is linear (r=0.999) between 30 and 3,360pg/ml of vIL-6. hIL-6 is not recognized in this vIL-6 ELISA. FIG. 6Bshows the detection of hIL-6, but not vIL-6, by a hIL-6-specific ELISA.

FIG. 7 is a graph showing the detection of vIL-6, hIL-6 and HIV RNA inserial serum samples from an HIV-positive patient with Castleman'sdisease. Day 1 denotes the day in which prednisone treatment wasinitiated. The lower limit of ELISA sensitivity in serum was calculatedto be 300 pg/mL of vIL-6 and 1.0 pg/mL of hIL-6. 3TC: lamivudine; d4T:stavudine; NFV: nelfinavir.

FIG. 8 is a chart documenting the detection of vIL-6 in sera from normalblood donors, HIV-positive patients with or without KS, and patientswith classic KS. The lower limit of ELISA sensitivity in serum sampleswas set at 300 pg/mL of vIL-6.

FIG. 9 is a set of schematic representations of vIL-6 structure. FIG. 9Ais a schematic diagram showing vIL-6 ABCD four helix bundle connected bypeptide loops. Site I, composed of the N-terminal region of the AB-loopand the C-terminal region of helix D, identifies the epitope recognizedby the vIL-6 neutralizing antibodies described here, and corresponds tothe presumed site where human IL-6 would interact with IL-6Rα. Site IIon helix A and C, and site III on the initial part of the AB-loop andhelix D represent binding surfaces to gp130. FIG. 9 B is a schematicdiagram of vIL-6 juxtaposed to gp130 in a tetrameric (2:2) signalingmodel based on the crystal structure of the complex. vIL-6 site II isoccupied by the D2D3 sites of one gp130 chain, and site III is occupiedby the D1 site of another gp130 chain. Site I of vIL-6, comprising theoutward helical face, is not occupied by gp130 and is stericallyaccessible for engagement by other molecules.

FIG. 10 is a set of graphs showing binding of human IL-6 or vIL-6 toimmobilized sgp130 using the biosensor system BIAcore 2000. sgp030 wasimmobilized at a concentration of 18.2 ng/mm2 on the CM5 biosensor chip.(A) Human IL-6 (50 μg/mL) alone or human IL-6 (50 μg/mL) plus sIL-6Rα(20 μg/mL), (B) MBPvIL-6 (50 μg/mL) alone or MBPvIL-6 (50 μg/mL) plussIL-6Rα (20 μg/mL) were passed over the sensor surface at a flow rate of10 μl/min in PBS. Reagents were incubated for 1 hour prior to assay. (C)Overlay of sensorgrams showing kinetic analysis of vIL-6 with sgp130.sgp130 was immobilized at a concentration of 1.5 ng/mm2. MBPvIL-6 (100,200, 400 and 800 μg/mL) was passed over the sensor surface. Control MBP(800 μg/mL) did not show detectable affinity for sgp130. Representativedata of four independent experiments are shown.

FIG. 11 is a schematic diagram of fusion protein mapping of anti-vIL-6mAbs. Schematic representation of deletion mutants of vIL-6 fusionproteins. Numbers above each box represent the amino acid positionsrelative to the start methionine. Restriction enzyme sites Aat II andEcoRI used to construct M3 and M5, respectively, are indicated. Thereactivity of each mAbs to each fusion protein was determined by ELISA.The reactivity of each mAb to each fusion protein is shown as positive(+) when the absorbance (A_(405/550)) was significantly higher (0.7absorbance units) than the background or negative (−) when theabsorbance was similar (within 0.05 absorbance units) to the background.The black box represents the putative vIL-6 signal peptide portion(Neipel et al., J Virol 1997; 71:839–842). The dotted box denotes a 13aa peptide of vIL-6 that is recognized by 4 neutralizing mAbs againstvIL-6. The bold characters indicate the second conserved cysteineresidues. vIL-6 sites I, II and III are defined based on the crystalstructure of the vIL-6/gp130 complex (Chow et al., Science 2001;291:2150–2155).

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The following definitions and methods are provided to better define thepresent invention, and to guide those of ordinary skill in the art inthe practice of the present invention. As used herein and in theappended claims, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the antibody” includes reference to one or moreantibodies, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs.

Ameliorate: A lessening of the detrimental effect of the KSHV-associateddisorder in a subject receiving therapy, such as a decrease in anyparameter of the disease, including any symptoms of the disorder.

Complex (complexed): Two proteins, or fragments or derivatives thereof,are said to form a complex when they measurably associate with eachother in a specific manner. Such association can be measured in any ofvarious ways, both direct and indirect. Direct methods may includeco-migration in non-denaturing fractionation conditions, for instance.Indirect measurements of association will depend on secondary effectscaused by the association of the two proteins or protein domains. Forinstance, the formation of a complex between a protein and an antibodymay be demonstrated by the antibody-specific inhibition of some functionof the target protein. In the case of vIL-6, the formation of a complexbetween vIL-6 and a specific binding agent (e.g. a neutralizingantibody) for this protein can be measured by determining the degree towhich the antibody inhibits an activity of vIL-6. Assays for vIL-6activity are discussed further below.

Diagnostically effective: The amount of detectably labeled specificbinding agent (e.g. a neutralizing antibody that binds vIL-6) that, whenadministered or utilized, is in sufficient quantity to enable detectionof vIL-6.

ELISA: Enzyme-linked immunosorbent assay. A form of quantitativeimmunoassay based on the use of antibodies (or antigens) that are linkedto an insoluble carrier surface, which is then used to capture therelevant antigen (or antibody) in the test solution. Theantigen-antibody complex is then detected by measuring the activity ofan appropriate enzyme that had previously been covalently attached tothe antigen (or antibody).

Epitope: Any antigenic determinant on an antigen to which the paratopeof an antibody binds. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

Gp130: A transmembrane glycoprotein with a length of 918 amino acids,including an intracellular domain of 277 amino acids, that is a subunitconstituent related to several cytokine receptors including the IL-6,IL-11, LIF, oncostatin M, CNTF cytokine receptors (see Gearing G P etal., Science 255: 1434–7, 1992; Hibi M. et al., Cell 63: 1149–57, 1990).Cytokines that share the gp130 subunits are sometimes referred to as the“IL-6 type family of cytokines.” Gp130 is one component of the receptorthat binds vIL-6.

Gp130 participates in the formation of high-affinity receptors for thesecytokines by binding to low affinity receptor chains. Accordingly, gp130has been called an “affinity converter.” IL-6 binding to a cytokinereceptor leads to the dimerization of gp130 and theactivation/association of a family of tyrosine kinases (the Januskinases) as the first step of intracellular signal transduction.

Using the structure of the growth hormone/growth hormone receptorcomplex as a paradigm for cytokine receptor complex assembly, IL-6-typecytokines are believed to have three topologically discrete sites ofinteractions with their receptors. Site I, if used, is always engaged bya non-signaling receptor: IL-6Rα, IL-11R or ciliary neurotrophic factorreceptor. Site II is always engaged by gp130, and site III by a secondsignaling receptor gp130, OSMR or LIFR (Bravo et al., EMBO J 2000;19:2399–2411). Within gp130, three binding epitopes have been identifiedas critical to its activation by human IL-6/IL-6Rα: one epitope involvesthe Ig-like domain (D1); another epitope is located in the cytokinebinding module (D2D3); and the other is located in the membrane-proximalextracellular domains (D4D5D6) (Kurth et al., J Immunol 2000;164:273–282).

IL-6: A cytokine produced by many different cell types (for review seeAkira S. et al., FASEB J. 4: 2860–7, 1990; Wolvekamp and Marquet,Immunology Let. 24: 1–9, 1990). The main sources in vivo are stimulatedmonocytes, fibroblasts, and endothelial cells, although macrophages,T-cells and B-lymphocytes, granulocytes, smooth muscle cells,eosinophils, chondrocytes, osteoblasts, mast cells, glial cells, andkeratinocytes also produce IL-6 after stimulation. Glioblastoma cellsconstitutively produce IL-6, and the factor can be detected also in thecerebrospinal fluid. Human milk also contains IL-6.

IL-6 is a protein of 185 amino acids glycosylated at positions 73 and172. It is synthesized as a precursor protein of 212 amino acids.Monocytes express at least five different molecular forms of IL-6 withmolecular masses of 21.5–28 kDa. The forms differ mainly bypost-translational alterations such as glycosylation andphosphorylation. The human IL-6 gene has a length of approximately 5 kband contains five exons. It maps to human chromosome 7p21-p14 betweenthe markers D7S135 and D7S370.

The crystal structure of IL-6 has identified an antiparallel four helixbundle (A, B, C and D) with a topology common to a number of othercytokines in the superfamily (Somers et al., EMBO J. 1997; 16:989–997).Extensive studies, including mutagenesis and mapping epitopes offunction-blocking or activating Ab, have demonstrated that the contactpoints of human IL-6 with its receptor complex are mediated by threedistinct sites named I, II and III (Brakenhoff et al., J Immunol 1990;145:561–568; Savino et al., EMBO J 1994; 13:5863–5870; Savino et al.,EMBO J 1994; 13:1357–1367; Kalai et al., Blood 1997; 89:1319–1333). SiteI, formed by the C-terminal part of helix D and in part by the AB-loop,interacts with IL-6Rα Site II, formed by a limited number of exposedresidues on helix A and helix C, and site III, formed by residues at thebeginning of helix D spatially flanked by residues in the initial partof the AB-loop, bind to gp130 (Somers et al., EMBO J 1997; 16:989–997).

Many assays have been developed to detect IL-6 (e.g. see Anderson etal., Kidney Internat, 40: 1110–7, 1991; De Groote et al., J. ImmunolMeth. 163: 259–67, 1993; Guba et al., Blood 80: 1190–8, 1992; Helle etal., J. Immunol Meth. 138: 47–56, 1991). For example, IL-6 can bedetected in bioassays employing IL-6 responsive cell lines (e.g. 7TD1,B9, CESS, KPMM2, KT-3, M1, MH60-BSF-2, MO7E, Mono Mac 6, NFS-60, PIL-6,SKW6-C14, T1165, XG-1). IL-6 can be assayed also by its activity as ahybridoma growth factor. Immunoassays and/or colorimetric tests are alsoavailable for IL-6. An alternative and entirely different detectionmethod is RT-PCR to detect IL-6 mRNA. In addition, an ELISA assay existsfor detecting the receptor-associated gp130 protein.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Kaposi's Sarcoma-Associated Herpesvirus (KSHV): A unique herpesvirus(also known as human herpes virus 8 or HHV-8) which has been identifiedin 100% of amplifiable samples of Kaposi's sarcoma in AIDS patients(patients infected with human immunodeficiency virus (HIV)) as well asin HIV-negative patients. The virus can be isolated from PBMC as well asKaposi' sarcoma tumor cells. HHV-8 also contains a considerable numberof viral genes that are similar to cellular genes in an ‘oncogeniccluster’ within the virus genome. The genes in the oncogenic cluster arebelieved to be involved in the development of malignancy (See Boshoff,Nature 391: 24–25, 1998). In addition to Kaposi's sarcoma, this virusmay play a role in the development of peripheral effusion lymphoma and aform of severe lymph node enlargement, called Castleman's disease.

In North America, probably less than 10% of the general population hasbeen infected with KSHV. The rates of KSHV infection in the generalpopulation of Mediterranean countries (Italy, Greece, Israel, SaudiArabia) are higher than in North America and Northern Europe. Adultpopulations in some portions of Africa have very high infection rates(>50%). It is believed that KSHV is transmitted by sexual and non-sexualroutes. Over 95% of persons who are healthy and infected with KSHV donot have symptoms. However, symptoms occur once an infected individualis immunosuppressed, such as by the use of pharmaceuticalimmunosuppressants (e.g. as used in transplant patients or to treatautoimmune disease), as a result of an HIV infection, or from the use ofchemotherapy. In addition, KSHV-related disease can also occur inpersons without obvious immunodeficiency, but this is rare and primarilyoccurs among elderly men.

The viral genome (140 kb) of KSHV has been sequenced completely. Theviral genome contains various open reading frames encoding proteins thatmimic the actions of cytokines or that are involved in cytokinesignaling.

KSHV-associated disorder: Any disorder associated with infection of asubject, such as a human subject, with KSHV. In one embodiment, thesubject is also infected with a human immunodeficiency virus (HIV). Inone specific, non-limiting example, the disorder is Kaposi's sarcoma. Inanother specific, non-limiting example, the disorder is Castleman'sdisease. In yet another specific, non-limiting example, the disorder isprimary effusion lymphoma (PEL).

KSHV viral IL-6 (vIL-6): The region of the genome of KSHV termed ORF-K2encodes a structural homologue of IL-6, termed viral IL-6 (vIL-6, seeU.S. Pat. No. 5,861,500, herein incorporated by reference). The vIL-6 ishomologous to cellular IL-6, and has a 47 percent amino acid similarityto human IL-6. The cysteine residues involved in disulfide bridging andthe highly conserved region involved in receptor binding are retained inthe viral gene product (see Burger, Blood 91:1858–1863, 1998; Molden etal., J. Biol. Chem. 272: 19625–19631, 1997; Neipel et al., J. Virol. 71:839–842, 1997). KSHV vIL-6 binds receptor components on the cellmembrane. One of the receptor components that binds KSHV vIL-6 is gp130.

vIL-6 is functionally active and can substitute for human IL-6 inseveral assays. For example, vIL-6 prevents mouse myeloma cellapoptosis. It also supports proliferation of tumor cells dependent onexogenous IL-6 for growth and/or survival, such as the human myelomacell line INA-6. vIL-6 is also functional in B9 proliferation bioassays.In addition, vIL-6 has been shown to activate the signaling pathwaysinvolving STAT proteins and Janus kinases via interactions with thegp130 signal transducing subunit. This interaction is independent of theIL-6 receptor alpha chain and may influence disease pathogenesis uponKSHV infection by interfering with signaling through gp130 in responseto native cytokines. vIL-6, as used herein, includes the amino acidsequence encoded by ORF-K2, conservative variants thereof, andbiologically active fragments thereof, wherein the vIL-6 amino acidsequence is active in an assay for vIL-6 activity.

Recently, the crystal structure of vIL-6 has revealed that vIL-6 shareswith other members of the IL-6 family the canonical up-up-down-down,ABCD four-helix bundle connected by peptide loops (Chow et al., Science2001; 291:2150–2155 (FIG. 9A). In that study, a soluble tetramericcomplex (2:2) of vIL-6 and the three NH2-terminal domains (D1D2D3) ofgp130 was crystallized in the absence of IL-6Rα. Based on thisvIL-6-gp130 crystal complex, the unused site I face of vIL-6, wherehuman IL-6 would interact with IL-6Rα, is not occupied by gp130 and issterically accessible for engagement by another molecule (FIG. 9B).

According to the crystallographic analysis of the vIL-6/sgp130 (D1D2D3)complex (Chow et al., Science 2001; 291:2150–2155), sequence alignmentof human and vIL-6 shows that contact residues seen in the structure ofthe vIL-6/gp130 complex are in the same positions as human IL-6-gp130contact residues previously mapped by mutagenesis (Simpson et al.,Protein Sci 1997; 6:929–955). In cell culture, the membrane-proximalextracellular part of gp130 (D4D5D6) is critical for vIL-6mediated-signaling, even though vIL-6 can form a tetrameric complex withgp130(D1D2D3) (Chow et al., Science 2001; 291:2150–2155). Further,although human and vIL-6 share an epitope in gp130 D2, vIL-6 does notappear to utilize an epitope in gp130 D3 that is critical to human IL-6function.

Lentivirus: Lentiviruses are characterized by long incubation periodsbetween infection of the host and the manifestation of clinical disease.Lentiviruses infect a wide variety of mammals, including humans,monkeys, sheep, goats, and horses. Includes for example retroviruses,such as immunodeficiency viruses, such as HIV-1, HIV-2, felineimmunodifficency virus (FIV), and simian immunodifficiency virus (SIV).

The human immunodeficiency virus (HIV) is the etiological agent of theacquired immunodeficiency syndrome (AIDS) and related disorders. Theexpression of the virus in infected persons is regulated to enable thevirus to evade the host's immune response. The HIV viruses (e.g. HIV-1and HIV-2), as well as the simian immunodeficiency virus (SIV), sharemany structural and regulatory genes such as gag, pol, env, tat, rev andnef (see Guyader et al., Nature 328:662–669, 1987). HIV has beenclassified as a lentivirus because it causes slow infection, and hasstructural properties in common with such viruses (Haase, Nature322:130–136, 1986).

Mammal: This term includes both human and non-human mammals. Similarly,the terms “subject,” “patient,” and “individual” includes human andveterinary subjects.

N-terminal region of a protein: Proteins are directional, and have anamino terminus (N-terminus), and a carboxy terminus (C-terminus). Theamino terminal (N-terminal) portion of a protein is the portion of theprotein near the N-terminus. In one embodiment, the N-terminal region isthe half of the protein near to the N-terminus. The N-terminal region ofa protein can include a receptor binding site of a protein, which is aregion of a protein that interacts with the receptor. It should be notedthat the C-terminal region of a protein can also include a receptorbinding site.

In one embodiment, the N-terminal region of vIL-6 is about half of thevIL-6 polypeptide near the N-terminus, or from about amino acid 1(numbering from the N-terminus) to about amino acid number 102 ofwild-type vIL-6. In one specific, non-limiting example, an N-terminalregion of vIL-6 includes about ten to about 20 amino acids of theN-terminal region of IL-6. In another specific, non-limiting example,the N-terminal region includes, but is not limited to, the vIL-6fragment inclusive of Asp⁸¹–Cys⁹³.

Neutralizing binding agents: A specific binding agent that is able tospecifically bind to a target protein in such a way as to inhibit thesubsequent biological functioning of that target protein is said to beneutralizing of that biological function. The inhibition can be at leasta 40%, 50%, 60%, 75%, 85%, 90%, or 100% inhibition of the biologicalfunction. In general, any protein that can perform this type of specificblocking activity is considered a neutralizing protein; antibodies aretherefore a specific class of neutralizing protein. The complex formedby binding of a neutralizing protein to a target protein is called aneutralizing complex.

In one embodiment, a neutralizing agent is an antibody. Antibodies thatbind to viral components and thereby prevent the binding of the viralcomponent to target host cells or a target protein and inhibit abiological function of the viral component are said to neutralize theviral component. Therefore, antibodies that bind to KSHV proteins andmeasurably reduce an activity of the virus are neutralizing antibodies.In one embodiment, a vIL-6 binding agent that binds the N-terminus ofKSHV vIL-6 is neutralizing. In one embodiment, neutralizing antibodiesbind a domain, such as about ten to about fifteen amino acids of thevIL-6 site I. In another embodiment, neutralizing antibodies bind about13 amino acids of the vIL-6 site I, such as a region of the C-terminalpart of the AB-loop and/or the beginning of helix B, although anyantigenic determinant of the N-terminus can be utilized. In onespecific, non-limiting example the 13 amino acids of the receptorbinding site have a sequence as set forth as SEQ ID NO: 3, or aconservative variant thereof. The use of an antigenic region on aprotein to provide epitopes appropriate for the natural or laboratorygeneration of neutralizing antibodies is known in the art (e.g. see WO98/36087; U.S. Pat. Nos. 5,843,454; 5,695,927; 5,643,756; and5,013,548).

Any assay for vIL-6 activity (see above) can be used to determine that avIL-6 specific binding agent is neutralizing. The assay can be either anin vivo or an in vitro assay. Specific, non-limiting examples, of assaysof use are an assay for the ability of vIL-6 to support theproliferation of mouse B9 cells. These assays are well known to one ofskill in the art. Briefly, B9 cells (approximately 2,000 cells per well)are cultured in a series of microwells (e.g. in a 96 microwell plate). Aseries of dilutions (decreasing concentrations, generally about 2 foldserial dilutions) of the sample are added to the wells, and the cellsare incubated with the sample (e.g. for 72 hours in a humidified 37° C.,5% CO₂ incubator). After incubation the cells are pulsed with[³H]thymidine (e.g. for 4 hours at 37° C.), and the amount of[³H]thymidine incorporated is measured (e.g. by liquid scintillationcounting). The measurement reflects the amount of biologically activeIL-6 present in the sample. In general, a standard is used that containsa known level of IL-6 activity to assess normalization of inter-assayvariation.

Other assays include an assay to determine the proliferation of thehuman myeloma cell line INA-6 (which is strictly dependent on exogenousIL-6 for growth and survival). Other assays of use are the stimulationthe growth of KSHV-infected PEL cells (Jones et al., Blood 94:2871,1999), the promotion of hematopoiesis (Aoki, et al., Blood 93:4034,1999), and the activation of STAT1, STAT3 and JAK1 phosphorylation(Molden et al., J. Bio.l Chem. 272:19625, 1997).

These assays are amenable to quantification and thus the percentstimulation or inhibition of targets can be measured. For example, inthe in vitro B9 cell assay, B9 cell proliferation in medium withoutvIL-6 is typically 10–20 fold lower than in medium containing vIL-6. AvIL-6 neutralizing agent may abolish completely or in part B9 cellgrowth stimulation induced by vIL-6. The degree of inhibition of vIL-6B9 cell growth stimulation by a neutralizing agent can be expressed as apercentage: 100% neutralization reflects a complete abrogation of B9cell stimulation by vIL-6 whereas a 50% inhibition reflects abrogationof 50% B9 cell stimulation induced by vIL-6.

Pharmaceutical agent or drug: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this invention are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the specific binding agents forvIL-6 herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polypeptide: any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation).

Specific binding agent: An agent that binds substantially only to adefined target. In one embodiment, a specific binding agent is aneutralizing binding agent. In one embodiment, a specific binding agentis neutralizing. In one embodiment, a vIL-6 specific binding agent is apolypeptide, such as a gp130 polypeptide, or a fragment thereof. Inanother embodiment, a KSHV vIL-6 specific binding agent includesanti-vIL-6 antibodies and other agents that bind substantially only tovIL-6.

In one embodiment, a specific binding agent that binds vIL-6 is aneutralizing binding agent (e.g. a neutralizing antibody). Neutralizingantibodies may be monoclonal or polyclonal antibodies that are specificfor vIL-6, and particularly its N-terminal domain, as well asimmunologically effective portions (“fragments”) thereof. In oneembodiment, a specific binding agent of the invention are monoclonalantibodies (or immunologically effective portions thereof) and may alsobe humanized monoclonal antibodies (or immunologically effectiveportions thereof). Immunologically effective portions of monoclonalantibodies include Fab, Fab, F(ab)₂, Fabc and Fv portions (for a review,see Better and Horowitz, Methods. Enzymol. 1989, 178:476–496).Anti-vIL-6 peptide antibodies may also be produced using standardprocedures described in a number of texts, including Antibodies, ALaboratory Manual by Harlow and Lane, Cold Spring Harbor Laboratory(1988), see below.

The determination that a particular agent binds substantially only tothe vIL-6 peptide may readily be made by using or adapting routineprocedures. One suitable in vitro assay makes use of the Westernblotting procedure (described in many standard texts, includingAntibodies, A Laboratory Manual by Harlow and Lane). Western blottingmay be used to determine that a given binding agent, such as ananti-vIL-6 antibody, binds substantially only to KSHV vIL-6.

Variants of Amino Acid Sequences: One of ordinary skill in the art willappreciate that a DNA sequence can be altered in numerous ways withoutaffecting the biological activity of the encoded protein, such as vIL-6.Conservative amino acid substitutions can preserve the functional andimmunologic identity of the encoded polypeptide. Conservativesubstitutions replace one amino acid with another amino acid that issimilar in size, hydrophobicity, etc. Examples of conservativesubstitutions are shown in Table 1 below.

TABLE 1 Original Residue Conservative Substitution Ala ser Arg lys Asngln, his Asp glu Cys ser Gln asn Glu asp Gly pro His asn; gln Ile leu,val Leu ile; val Lys arg; gln; glu Met leu; ile Phe met; leu; tyr Serthr Thr ser Trp tyr Tyr trp; phe Val ile; leu

More substantial changes in protein function may be obtained byselecting amino acid substitutions that are less conservative than thoselisted in Table 4. Such changes include changing residues that differmore significantly in their effect on maintaining polypeptide backbonestructure (e.g., sheet or helical conformation) near the substitution,charge or hydrophobicity of the molecule at the target site, or bulk ofa specific side chain. The following substitutions are generallyexpected to produce the greatest changes in protein properties: (a) ahydrophilic residue (e.g., seryl or threonyl) is substituted for (or by)a hydrophobic residue (e.g., leucyl, isoleucyl, phenylalanyl, valyl oralanyl); (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain (e.g.,lysyl, arginyl, or histadyl) is substituted for (or by) anelectronegative residue (e.g., glutamyl or aspartyl); or (d) a residuehaving a bulky side chain (e.g. phenylalanine) is substituted for (orby) one lacking a side chain (e.g., glycine).

Variant encoding sequences may be produced by standard DNA mutagenesistechniques, for example, M13 primer mutagenesis. Details of thesetechniques are provided in Sambrook et al. (In Molecular Cloning: ALaboratory Manual, CSHL, New York, 1989), Ch. 15. By the use of suchtechniques, variants may be created which differ in minor ways from thehuman sequences disclosed.

Alternatively, the coding region may be altered by taking advantage ofthe degeneracy of the genetic code to alter the coding sequence suchthat, while the nucleotide sequence is substantially altered, itnevertheless encodes a protein having an amino acid sequencesubstantially similar to the disclosed protein sequences. Based upon thedegeneracy of the genetic code, variant DNA molecules may be derivedfrom cDNA and gene sequences using standard DNA mutagenesis techniquesas described above, or by synthesis of DNA sequences.

The immunologic identity of the protein may be assessed by determiningwhether it binds to a specific binding agent (e.g., a neutralizingantibody that binds vIL-6); a variant that is recognized by such anantibody is immunologically conserved. Any cDNA sequence variant willpreferably introduce no more than 20, and preferably fewer than 10 aminoacid substitutions into the encoded polypeptide. Variant amino acidsequences may, for example, be 80, 90 or even 95% identical to thenative amino acid sequence.

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS Specific Binding Agentsfor vIL-6

Kaposi's sarcoma-associated herpesvirus interleukin-6 can be used toproduce neutralizing agents that bind vIL-6. In one embodiment, theseagents are antibodies which are immunoreactive or bind to epitopes orvariants of vIL-6 and inhibit a biological function of vIL-6. Antibodieswhich consist essentially of pooled monoclonal antibodies with differentepitopic specificities, polyclonal antibodies as well as distinctmonoclonal antibody preparations are provided, as long as the antibodiesinterfere with a biological activity of vIL-6.

In one embodiment, the specific binding agent binds the N-terminus ofKSHV vIL-6, and neutralizes an activity of vIL-6. In another embodiment,the specific binding agent binds a portion of vIL-6 in a region of theC-terminal part of the AB-loop and/or the beginning of helix-B, and thusneutralizes an activity of vIL-6. In one specific, non-limiting example,the specific binding agent binds about ten to about fifteen amino acids,although the specific binding agent can bind a number of amino acids inthe region. In one embodiment, the monoclonal antibody induces aconformational change that affects the binding of vIL-6 to gp130.

In one embodiment, the specific binding agent binds a region of vIL-6 inthe N-terminal portion of vIL-6. In one specific, non-limiting example,the specific binding agent binds about ten to about twenty amino acidsof the N-terminal portion of vIL-6. For example, the specific bindingagent binds about thirteen amino acids of the N-terminal portion ofvIL-6, such as DHCGLIGFNETSC (SEQ ID NO: 3), or a conservative variantthereof, and wherein binding of the specific binding agent neutralizesan activity of vIL-6. In a specific non-limiting examples, themonoclonal antibody specifically binds a domain within the vIL-6 site 1,such as a region of the C-terminal part of the AB-loop and/or thebeginning of helix-B.

The specific binding agent can be a polypeptide or a fragment thereof, achemical or pharmaceutical compound, or an antibody.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, for example, Green et al., “Production of PolyclonalAntisera,” in: Immunochemical Protocols pages 1–5, Manson, ed., HumanaPress 1992; Coligan et al., “Production of Polyclonal Antisera inRabbits, Rats, Mice and Hamsters,” in: Current Protocols in Immunology,section 2.4.1, 1992, which are hereby incorporated by reference.

The preparation of monoclonal antibodies likewise is conventional. See,for example, Kohler & Milstein, Nature 256:495, 1975; Coligan et al.,sections 2.5.1–2.6.7; and Harlow et al., in: Antibodies: a LaboratoryManual, page 726, Cold Spring Harbor Pub., 1988, which are herebyincorporated by reference. Briefly, monoclonal antibodies can beobtained by injecting mice with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B lymphocytes, fusing the Blymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones that produce antibodies to theantigen, and isolating the antibodies from the hybridoma cultures.Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See,e.g., Coligan et al., sections 2.7.1–2.7.12 and sections 2.9.1–2.9.3;Barnes et al., “Purification of Immunoglobulin G (IgG),” in: Methods inMolecular Biology, Vol. 10, pages 79–104, Humana Press, 1992.

Methods of in vitro and in vivo multiplication of monoclonal antibodiesare well known to those skilled in the art. Multiplication in vitro maybe carried out in suitable culture media such as Dulbecco's ModifiedEagle Medium or RPMI 1640 medium, optionally supplemented by a mammalianserum such as fetal calf serum or trace elements and growth-sustainingsupplements such as normal mouse peritoneal exudate cells, spleen cells,thymocytes or bone marrow macrophages. Production in vitro providesrelatively pure antibody preparations and allows scale-up to yield largeamounts of the desired antibodies. Large scale hybridoma cultivation canbe carried out by homogenous suspension culture in an airlift reactor,in a continuous stirrer reactor, or in immobilized or entrapped cellculture. Multiplication in vivo may be carried out by injecting cellclones into mammals histocompatible with the parent cells, e.g.,syngeneic mice, to cause growth of antibody-producing tumors.Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection. After oneto three weeks, the desired monoclonal antibody is recovered from thebody fluid of the animal.

Therapeutic applications for antibodies disclosed herein are also partof the present invention. Thus, antibodies of the present invention mayalso be derived from subhuman primate antibody. General techniques forraising therapeutically useful antibodies in baboons can be found, forexample, in Goldenberg et al., International Patent Publication WO91/11465, 1991, and Losman et al., Int. J. Cancer 46:310, 1990).

Alternatively, a therapeutically useful anti-vIL-6 neutralizing antibodymay be derived from a “humanized” monoclonal antibody. Humanizedmonoclonal antibodies are produced by transferring mouse complementaritydetermining regions from heavy and light variable chains of the mouseimmunoglobulin into a human variable domain, and then substituting humanresidues in the framework regions of the murine counterparts. The use ofantibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions. General techniques for cloning murine immunoglobulinvariable domains are described, for example, by Orlandi et al., Proc.Natl. Acad. Sci. USA 86:3833, 1989). Techniques for producing humanizedmonoclonal antibodies are described, for example, by Jones et al.,Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyenet al., Science 239:1534, 1988; Carter et al., Proc. Natl Acad. Sci. USA89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer etal., J. Immunol. 150:2844, 1993.

Antibodies of the invention also may be derived from human antibodyfragments isolated from a combinatorial immunoglobulin library. See, forexample, Barbas et al., in: Methods: a Companion to Methods inEnzymology, Vol. 2, page 119, 1991; Winter et al., Ann. Rev. Immunol.12:433, 1994, which are hereby incorporated by reference. Cloning andexpression vectors that are useful for producing a human immunoglobulinphage library can be obtained, for example, from STRATAGENE CloningSystems (La Jolla, Calif.).

In addition, antibodies may be derived from a human monoclonal antibody.Such antibodies are obtained from transgenic mice that have been“engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994;and Taylor et al., Int. Immunol. 6:579, 1994.

Neutralizing antibodies used include intact molecules as well asfragments thereof, such as Fab, F(ab′)₂, and Fv which are capable ofbinding the epitopic determinant. These antibody fragments retain someability to selectively bind with its antigen or receptor and are definedas follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody (“SCA”), defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art (See for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988, incorporated herein by reference). Antibodyfragments can be prepared by proteolytic hydrolysis of the antibody orby expression in E. coli of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)₂. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and an Fcfragment directly. These methods are described, for example, byGoldenberg, U.S. Pat. No. 4,036,945 and No. 4,331,647, and referencescontained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230,1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods inEnzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al.at sections 2.8.1–2.8.10 and 2.10.1–2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association may be noncovalent, as described in Inbar etal., Proc. Natl. Acad. Sci. USA 69:2659, 1972. Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu,supra. Preferably, the Fv fragments comprise V_(H) and V_(L) chainsconnected by a peptide linker. These single-chain antigen bindingproteins (sFv) are prepared by constructing a structural gene comprisingDNA sequences encoding the V_(H) and V_(L) domains connected by anoligonucleotide. The structural gene is inserted into an expressionvector, which is subsequently introduced into a host cell such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingsFvs are described, for example, by Whitlow et al., Methods: a Companionto Methods in Enzymology, Vol. 2, page 97, 1991; Bird er al., Science242:423–426, 1988; Ladner et al., U.S. Pat. No. 4,946,778; Pack et al.,Bio/Technology 11:1271–77, 1993; and Sandhu, supra.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.Methods: a Companion to Methods in Enzymology, Vol. 2, page 106, 199).

Antibodies which bind to vIL-6, a variant thereof, or a biologicallyactive fragment thereof, can be prepared using an intact polypeptide orfragments containing small peptides of interest as the immunizingantigen. In one embodiment, the N-terminus of vIL-6 is utilized toprepare antibodies that bind to vIL-6. In another embodiment, apolypeptide that encompasses a region of the C-terminal part of theAB-loop and the beginning of helix-B is utilized. In one specific,non-limiting example, about ten to about twenty amino acids areutilized, although any antigenic determinant of the N-terminus can beutilized. In one specific, non-limiting example the thirteen amino acidsare utilized that have a sequence as set forth as SEQ ID NO: 3, or aconservative variant thereof.

The polypeptide or a peptide used to immunize an animal can be derivedfrom translated cDNA or chemical synthesis which can be conjugated to acarrier protein, if desired. Such commonly used carriers which arechemically coupled to the peptide include keyhole limpet hemocyanin(KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.The coupled peptide is then used to immunize the animal (e.g., a mouse,a rat, or a rabbit).

If desired, polyclonal or monoclonal antibodies can be further purified,for example, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Various techniques are common in the immunology arts for purificationand/or concentration of polyclonal antibodies, as well as monoclonalantibodies (See for example, Coligan et al., Unit 9, Current Protocolsin Immunology, Wiley Interscience, 1991).

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

Cell-Free Assay for Testing the Efficacy of a Neutralizing Agent

In order to test the efficacy of an agent for treating or detecting aKSHV associated disorder, a cell free system has been developed. Thiscell free system includes isolated vIL-6 and a receptor component thatbinds vIL-6 (e.g. gp130). The binding of vIL-6 and the vIL-6 receptorcomponent in the presence of the agent is compared to binding of vIL-6to the vIL-6 receptor component in the absence of the agent. A decreasein the binding of vIL-6 to the receptor component in the presence of theagent indicates that the agent is neutralizing, and thus is effectivefor treating a KSHV-associated disorder.

In one embodiment, the method includes incubating agents and a samplecontaining vIL-6 and a receptor component that binds vIL-6 underconditions sufficient to allow the components to interact, and measuringthe effect of the compound on the interaction of the receptor componentand vIL-6. Agents which specifically bind vIL-6 and affect. theinteraction of the receptor component and vIL-6 include peptides,polypeptides, chemical compounds and biological agents. In one specific,non-limiting example, the receptor component is gp130. In anotherspecific, non-limiting example, the agent is a polypeptide fragment ofgp130. Antiviral, immunosuppressive, and chemotherapeutic compounds canbe tested using the method of the invention.

Incubating includes any conditions that allow contact between the testagent, vIL-6 and the receptor component (e.g. gp130). “Contacting”includes in solution and solid phase. The test compound may also be acombinatorial library for screening a plurality of compounds. Compoundsidentified in the method of the invention can be further evaluated,detected, cloned, sequenced, and the like, either in solution of afterbinding to a solid support, by any method usually applied to thedetection of a specific DNA sequence, such as PCR, oligomer restriction(Saiki et al., Bio/Technology 3:1008–1012, 1985), allele-specificoligonucleotide (ASO) probe analysis (Conner et al., Proc. Natl. Acad.Sci. USA 80:278, 1983), oligonucleotide ligation assays (OLAs)(Landegren et al., Science 241:1077, 1988), and the like. Moleculartechniques for DNA analysis have been reviewed (Landegren et al.,Science 242:229–237, 1988).

The binding affinities of agents which affect the interaction of thereceptor component with vIL-6 can also be determined. In these assays, alabeled ligand is employed. A number of labels have been indicatedpreviously (e.g., radiolabels, fluorescence labels, among others) to beof use. The candidate compound is added in an appropriate bufferedmedium. After an incubation to ensure that binding has occurred, thesurface may be washed free of any nonspecifically bound components ofthe assay medium, particularly any nonspecifically bound labeled ligand,and any label bound to the surface determined. The label may bequantitatively measured. By using standards, the relative bindingaffinity of a candidate compound can be determined.

Detection or Treatment of a KSHV Associated Disorder

The specific binding agents (e.g. neutralizing antibodies) that bindvIL-6 can be used to detect or treat a KSHV-associated disorder or aKSHV-related disorder. The term “KSHV-associated disorder” denotes anydisorder associated with KHSV infection, including, but not limited to,Kaposi's sarcoma, primary effusion lymphoma (PEL), and Castleman'sdisease.

The specific binding agents that neutralize a biological activity ofvIL-6 can be used to determine the prognosis of a KSHV-associateddisorder. They can also be useful in guiding choices between differenttreatment regimens in patients with KSHV-associated or -relateddisorders. The “prognosis” is a forecast as to the probable outcome ofan attack of a disease; the prospect as to recovery from a disorder asindicated by the nature and symptoms of the case. In addition, thespecific binding agents for vIL-6 may be used to identify or treatindividuals who are “at risk” of developing a KSHV-associated disorder.These individuals may be identified by a method of the invention fordetecting the presence or absence of KSHV vIL-6 or by any otherdiagnostic means, and/or may be treated with the specific binding agentthat neutralizes a biological activity of vIL-6, prior to the actualonset of the clinical appearance (any sign or symptom) of disorder.

An antibody or other specific binding agent that neutralizes abiological activity of vIL-6 can be used to detect vIL-6 polypeptide insubject samples such as biological fluids, cells, tissues, or nucleicacid. Any specimen containing a detectable amount of antigen (vIL-6) canbe used. Examples of biological fluids of use with the invention areblood, serum, plasma, urine, mucous, and saliva. Tissue, cell samples,or extracts thereof can also be used with the subject invention. Thesamples can be obtained by many methods such as cellular aspiration, orby surgical removal of a biopsy sample.

The invention provides a method for detecting vIL-6, which comprisescontacting a specific binding agent that neutralizes a biologicalactivity of vIL-6 with a sample suspected of containing vIL-6, anddetecting binding of the specific binding agent to vIL-6 in the sample.The specific binding agent that neutralizes a biological activity ofvIL-6 is preferably labeled with a compound which allows detection ofbinding to vIL-6. The level of vIL-6 in the subject sample can becompared with the level in a sample not affected by the disease process.The sample not affected by the disease process can be taken from acontrol subject not affected by the disease process, or can be from acell line, or can be a blank control (medium).

The specific binding agents that neutralize a biological activity ofvIL-6 can be used in any subject in which it is desirable to administerin vitro or in vivo immunodiagnosis or immunotherapy. For example,neutralizing antibodies of the invention are suited for use, forexample, in immunoassays in which they can be utilized in liquid phaseor bound to a solid phase carrier. In addition, the antibodies in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of immunoassays which can utilize antibodies of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay (e.g. ELISA). Those of skillin the art will know, or can readily discern, an appropriate immunoassayformat without undue experimentation.

The neutralizing antibodies of the invention can be bound to manydifferent carriers, both soluble and insoluble, and used to detect thepresence of vIL-6. Examples of well-known carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses andmagnetite. Those skilled in the art will know of other suitable carriersfor binding antibodies, or will be able to ascertain such, using routineexperimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds,phosphorescent compounds, and bioluminescent compounds. Those ofordinary skill in the art will know of other suitable labels for bindingto the antibody, or will be able to ascertain such, using routineexperimentation.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyl, puridoxal, and fluorescein, which canreact with specific antihapten antibodies.

In using the antibodies that specifically bind vIL-6 and neutralize abiological activity of vIL-6 for the in vitro or in vivo detection ofantigen, the detectably labeled antibody is given a dose which isdiagnostically effective. The antibodies that specifically bind vIL-6and neutralize an activity of vIL-6 can be used in vitro and in vivo tomonitor the course of amelioration of a KSHV-associated disorder in asubject. Thus, for example, by measuring the changes in theconcentration of vIL-6 in various body fluids, it would be possible todetermine whether a particular therapeutic regimen aimed at amelioratingthe KSHV-associated disease is effective. The term “ameliorate” denotesa lessening of the detrimental effect of the KSHV-associated disease inthe subject receiving therapy.

Kits

The specific binding agents are ideally suited for the preparation of akit. Such a kit may comprise a carrier such as a box or a bag made ofany material (e.g., plastic or paper) containing one or more containerssuch as vials, tubes, and the like, each of the container meanscomprising one of the separate elements to be used in the method. One ofthe containers can include a specific binding agent that neutralizes anactivity of vIL-6 which is or can be detectably labeled. In onespecific, non-limiting example, the binding agent is an antibody, orspecific fragment thereof, that neutralizes an activity of vIL-6. Forexample, an antibody that neutralizes an activity of vIL-6 can beincluded in a kit and used for examining the presence of vIL-6 in asample, as well as a control sample for comparison. Thus, the binding ofthe neutralizing antibody with a test sample from a subject of interestcan then be compared with the binding of the neutralizing antibody withthe control included in the kit. The relative degree of binding to thesample as compared to the control indicates infection with KSHV, or thelikelihood for an subject developing a KSHV-associated disorder.

The kit can also contain a container including a second antibody whichbinds to the antibody that specifically binds and neutralizes anactivity of vIL-6. The second antibody can be directly labeled.Alternatively, the kit may also be a container including a reportermolecule, such as avidin or steptavin, bound to a molecule such as anenzymatic, fluorescent, or radionucleotide label to identify thedirectly labeled second antibody.

The kit can also contain directions for use. This includes writteninstructions or instructions in an electronic format, such as on adiskette or CD-rom disk.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using this description, utilize the present invention to itsfullest extent. The following examples are illustrative only, and notlimiting of the remainder of the disclosure in any way whatsoever.

Example 1 Materials and Methods

Preparation of recombinant vIL-6 and its deletion mutants: RecombinantvIL-6 was prepared as a fusion protein that has a factor Xa cleavagesite between the amino terminal tag of maltose binding protein (MBP) andamino acids 22–204 of vIL-6 (MBPvIL-6) (Aoki et al., Blood 93:4034,1999). Deletion mutants of vIL-6 were prepared as follows: vIL-6 M1 andM2 were constructed by inserting a stop codon at positions 37 and 81,respectively. After denaturing, pMALvIL-6 was annealed with thefollowing mutagenesis oligonucleotides:

M1: ATTTAGATCTTCAATTGGATGCTA; (SEQ ID NO:5) M2:ACTTAGATCTGCGGGTTAATAGGA; (SEQ ID NO:6)that encode a restriction enzyme site for Bgl II as well as stop codons,and mutant strands were synthesized using GeneEditor in vitrosite-directed mutagenesis system (Promega, Madison, Wis.). Aftertransformation, positive colonies were screened by GeneEditor and Bgl IIdigestion. vIL-6 M3 and M5 were constructed by restriction enzymedigestion of pMALvIL-6 with Aat II and EcoRI, respectively, followed byligation. To construct expression vectors for vIL-6 M4, M6 and M7,fragments including the C-terminus of vIL-6 were amplified using thefollowing oligonucleotide primers: M4-Bam HI:ACTGGATCCCTTAAAAAGCTCGCCGAT (SEQ ID NO:7); M6-Bam HI:TTTGGATCCTTAACGACGGAGTTTGGA (SEQ ID NO:8); M7-Bam HI:ACGGGATCCAGTCCACCCAAA TTTGAC (SEQ ID NO:9) and vIL-6-3′-HindCCCAAGCTTATTACTTATCGTGGACGT(SEQ ID NO:10). After digestion with Bam HIand Hind III, the PCR products were ligated into pMAL-c2 (New EnglandBioLabs, Beverly, Mass.). All recombinant protein-expressing clones wereanalyzed by DNA sequence analysis, and expressed in Escherichia colistrain DH5α (Life Technologies, Gaithersburg, Md.). Large-scaleproduction and affinity purification of fusion proteins were performedaccording to the manufacturer's instructions.

Generation of mAbs against vIL-6: To generate mAbs against vIL-6, micewere immunized with MBPvIL-6. After boosting, splenocytes were obtainedfor hybridoma production by standard procedures. Hybridomas werescreened by enzyme-linked immunosorbent assay (ELISA) and Westernblotting against recombinant vIL-6. Following bulk culture, mAbs werepurified from culture supernatants using protein G columns (Pierce,Rockford, Ill.). The isotype of each mAb was determined using MouseTyper sub-isotyping kit (Bio-Rad, Hercules, Calif.), according to themanufacturer's instructions.

Western blotting: Immunoblotting of vIL-6 was performed as describedpreviously (Jones et al, Blood 94:2871, 1999). Briefly, 100 ng ofMBPvIL-6 cleaved with factor Xa (New England BioLabs), recombinant hIL-6(a kind gift from Sandoz Pharmaceuticals) and recombinant mIL-6(PeproTech, Rocky Hill, N.J.) were loaded onto each well of 10–20%tricine-SDS gel (NOVEX, San Diego, Calif.) after boiling for 10 minutes.The separated proteins were transferred onto polyvinylidene fluoridemembranes (Immobilon-P; Millipore, Bedford, Mass.). Immunostaining wasperformed using rabbit polyclonal Abs against MBPvIL-6 (Aoki et al.,Blood. 93:4034, 1999), mIL-6 (PeproTech) or mouse anti-vIL-6 in Absfollowed by incubation with HRP-conjugated anti-mouse or rabbit IgG Abs(Amersham, Piscataway, N.J.). Immunocomplexes were visualized using thechemiluminescence detection system (Amersham).

Mapping the epitopes recognized by the monoclonal antibodies: Fulllength and deletion mutants of vIL-6 fusion proteins were immobilizedonto 96-well plates (Immulon 4HBX; Dynex Technologies, Chantilly, Va.)in phosphate buffered saline (PBS) by overnight incubation at 4° C.After blocking nonspecific binding with SuperBlock (Pierce), a standardELISA protocol was followed (Kellam et al., J. Virol. 73:5149, 1999;Jones et al., J Exp Med. 182:1213, 1995) using appropriate dilutions ofanti-vIL-6 Ab and a 1:500 dilution of goat anti-mouse polyvalent Igconjugated to alkaline phosphatase (Sigma, St Louis, Mo.). Reactionswere visualized by using p-nitrophenyl phosphate (Sigma), and plateswere read at 405 nm with λ correction at 550 nm using a microplatereader.

vIL-6 bioassays: The B9 cell proliferation assay was performedessentially as described (Jones et al., J Exp Med. 182:1213, 1995). B9cells (2×10³ cells/well) were incubated in 96-well plates (200 μl/well)with MBPvIL-6 (100 ng/ml) and anti-vIL-6 mAbs (0 to 10 μg/ml) for 72 hat 37° C., including a 6-h terminal pulse with 1 μCi/well of[³H]-thymidine (Amersham, Arlington Heights, Ill.). [³H]-thymidineincorporation was determined after cell harvesting onto glass fiberfilters. The SKW6.4 IgM secretion assay was performed essentially asdescribed (Peppard et al., Biol. Chem. 271:7281, 1996). SKW6.4 cells(1×10⁴ cells/well) were incubated in 96-well plates (200 μl/well) withMBPvIL-6 (0 to 4 μg/ml) or hIL-6 (0 to 20 ng/ml) with or withoutanti-vIL-6 mAbs (0 to 10 μg/ml) for 96 h at 37° C. IgM levels in theculture supernatants were measured using a human IgM ELISA quantitationkit (Bethyl Laboratories Inc, Montgomery, Tex.), according to themanufacturer's instructions. This ELISA does not detect murine IgG.

ELISA-based binding assay: The binding of vIL-6 to IL-6Rα was analyzedby ELISA (FIG. 6) as described previously with some modifications (JBiol. Chem. 273:21374, 1998). Purified recombinant human sIL-6Rα (R&DSystems, Minneapolis, Minn.) and human sgp130 (R&D Systems) wereimmobilized on ELISA plate wells (Immunol 4HBX) at 5 μg/ml in PBS. Afterblocking the plate with SuperBlock, MBPvIL-6 or hIL-6 were applied at 50ng/ml in 1% BSA/PBS, and incubated for 5 h at room temperature. Boundprotein was detected with polyclonal rabbit Abs directed against vIL-6or hIL-6 (Pepro Tech) at 1 μg/ml, followed by an HRP-conjugatedanti-rabbit IgG (Bio-Rad) at 1:3000 in PBS/0.05% Tween 20. Reactionswere visualized by using tetramethoxybenzene peroxidase substrate(Kirkegaard & Perry Laboratories, Gaithersburg, Md.), followed by 2NH2SO4. The interference of vIL-6 binding to sgp130 by mAbs was analyzedin a similar manner. Biotinylated MBPvIL-6 (Jones et al., Blood 94:4034,1999) was incubated at 50 ng/ml in 1% BSA/PBS with anti-vIL-6 mAbs orisotype control murine IgG1 (20 μg/ml) overnight at 4° C. The mixturewas applied to the plates coated with sgp130 (2 μg/ml) after SuperBlocktreatment. The protein complex was detected by streptavidin-HRP (1:1000;Kirkegaard & Perry Laboratories), and the peroxidase activity wasvisualized by tetramethoxybenzene peroxidase substrate, followed by 2NH₂SO₄. Plates were read at 450 nm with λ correction at 630 m using amicroplate reader.

Surface plasmon resonance: Biospecific interaction analysis wasmonitored by surface plasmon resonance using BIAcore 2000 system(Biacore AB, Uppsala, Sweden). sgp130-Fc (R&D systems), a fusion proteinof sgp130 and human IgG Fc, was immobilized onto the CM5(carboxymethylated dextran matrix) sensor chip using the amine couplingkit (BIAcore AB), and unreactive groups on the chip were blocked byethanolamine according to the manufacturer's instructions. A continuousflow (10 μL/mL) of human IL-6 or MBPvIL-6 onto immobilized sgp130-Fc wasmonitored by passing the analytes across the sensor chip. In parallelexperiments, sIL-6Rαwas incubated with human IL-6 and MBPvIL-6 prior toassay. In comparative experiments, a continuous flow (20 μL/mL) ofsIL-6Rαonto immobilized human IL-6 or MBPvIL-6 was monitored in the samemanner. The sensor surface was regenerated between assays by 30 secondstreatment of 10 mM Glycine pH 2.2. BIAevaluation 3.0 software (BIAcoreAB) was used for all the interaction analyses. The kinetics parameterwas calculated according to a 1:1 Langmuir binding model (A+B

AB) by direct fitting of ligand binding sensorgrams at multipleconcentrations. The dissociation equilibrium constant is defined asdissociation constant (K_(d))=dissociation rate constant(k_(d))/association rate constant (k_(a)). The K_(d) was also determinedby Scatchard analysis of equilibrium-state data obtained by a continuousflow (1 μL/mL) of MBPvIL-6 (50–800 μg/L) onto immobilized sgp130-Fc,according to previously published methods (Ward et al., Biochemistry1995; 34:2901–2907).

Example 2 Binding of vIL-6 to sgp130 in the Absence of sIL-6Rα

It is known that gp130 is involved in the signal transduction eventswhich follow cell stimulation with vIL-6 (Nicholas et al., Nat Med.3:287, 1997; Molden et al., J Biol Chem. 272:19625, 1997; Hideshima etal., Clin Cancer Res. 6:1180, 2000; Burger et al., Blood 91:1858, 1998;Wan et al., J Virol. 73:8268, 1999; Gage et al., AIDS 13:1851, 1999;Mullberg et al., Immunol. 164:4672, 2000). In contrast, the usage ofIL-6Rα by vIL-6 is still controversial. Soluble forms of IL-6Rα andgp130, lacking the transmembrane and cytoplasmic regions, have beendetected in human sera and body fluids (Peters et al., Blood 92:3495,1998; Murakami-Mori et al., Int Immunol. 8:595, 1996) and have beenproduced as recombinant proteins. sIL-6Rα, when complexed with IL-6,acts agonistically on cells that express gp130, whereas sgp130 acts asan antagonist for IL-6 signaling (Taga et al., An. Rev. Immunol. 15:797,1997; Peters et al., Blood 92:3495, 1998). It was investigated whethervIL-6 could bind to purified sIL-6Rα and sgp130 in a cell-free system.

Purified human sIL-6Rα and/or sgp130 were immobilized onto a 96-welldish, and vIL-6 binding was assayed by indirect ELISA. As expected,hIL-6 bound to sIL-6Rα but not to sgp130 alone. When sIL-6Rα and sgp130were first incubated and then immobilized onto wells, the binding ofhIL-6 was slightly reduced (FIG. 1). By contrast, MBPvIL-6 bound towells coated with sgp130 but not to wells coated with sIL-6Rα alone. Thebinding of vIL-6 to sgp130 was minimally affected by pre-incubation ofsgp130 with sIL-6Rα. In parallel experiments, sIL-6Rα and sgp130immobilized on a plate failed to bind to MBP.

Lack of IL-6Rα contribution to vIL-6/gp130 binding was further confirmedby surface plasmon resonance. The CM5 sensor chip immobilized withsgp130 was used for equilibrium binding analysis on the BIAcore system.Human IL-6 showed strong binding to sgp130 only when preincubated withsIL-6Rα (FIG. 10A). In contrast, vIL-6 bound to sgp130 without sIL-6Rα,and vIL-6 preincubation with sIL-6Rα minimally increased bindingaffinity for sgp130 (FIG. 10B). In accordance with these observations,sIL-6Rα bound to the CM5 sensor chip immobilized with human IL-6 but notMBPvIL-6 even though MBPvIL-6 was used at up to 2.2-times higher amountthan that of human IL-6. A range of MBPvIL-6 concentrations was injectedover the immobilized surface of sgp130, and, as a control, MBP (FIG.10C). Control MBP showed minimal signal increase over base line. Bindingof MBPvIL-6 to sgp130 demonstrated concentration dependence andsaturability. Using a 1:1 Langmuir binding model, sensograms yielded ak_(a) of 980 1/Ms, k_(d) of 2.2×10⁻³ 1/s and Kd of 2.2 μM. AdditionalBIAcore data of MBPvIL-6 binding to immobilized sgp130 at equilibriumwas analyzed by Scatchard analysis. Using this method, the Kd for thevIL-6-gp130 interaction was calculated at 2.5 μM, which is comparable tothe Kd obtained by the 1:1 Langmuir binding model. Previously, theaffinity of human IL-6/sIL-6Rα complex for gp130 yielded a Kd of 1.7 nM.Thus, the binding affinity of vIL-6 for sgp130 is 1000-fold lower thanthat of human IL-6/sIL-6Rα complex to gp130. Together, these experimentsdemonstrate that vIL-6 can bind to gp130 but not IL-6Rα.

Example 3 Generation of mAbs Against KSHV vIL-6: Epitope Mapping

To investigate further vIL-6 interactions with its receptor,vIL-6-specific mAbs were generated. Mice were immunized with the fusionprotein of vIL-6 and MBP, MBPvIL-6 that was purified from Escherichiacoli (Aoki et al., Blood 93:4034, 1999). Hybridomas were screened byELISA against vIL-6 protein that was obtained from supernatants ofNIH3T3 cells stably transfected with vIL-6 (Aoki et al., supra).Following a second screening, six anti-vIL-6 mAbs with IgG1 isotype wereselected. It was first determined if these Abs recognize vIL-6 byWestern blotting. All six mAb recognized vIL-6 protein that was obtainedby cleaving MBPvIL-6 with factor Xa (Jones et al., Blood 94:2871, 1999).These Abs also detected vIL-6 in cell lysates of the KSHV-positive PELcell line BCP-1 (Moore et al., Science 274:1739, 1996) but not in thecontrol KSHV-negative Daudi cells by Western blotting. Although vIL-6exhibits 25% amino acid identity to cellular IL-6, none of these mAbsrecognized either hIL-6 or mIL-6. A rabbit polyclonal antiserum raisedagainst vIL-6 recognized vIL-6 but not hIL-6 or mIL-6, whereas a rabbitpolyclonal antiserum raised against murine IL-6 (mIL-6) recognized mIL-6and hIL-6, but not vIL-6. These results suggest that the highlyimmunogenic epitopes on vIL-6 are distinct from those of cellular IL-6.

Example 4 An Assay for Neutralizing Activity of mAbs in IL-6-ResponsiveCell Lines

In order to assess if these mAbs neutralize vIL-6 bioactivities, a B9proliferation assay was utilized (FIG. 2). vIL-6 has previously beenshown to accelerate the proliferation of murine hybridoma B9 cells(Burger et al., Blood 91:1858, 1999; Aoki et al., Blood 93:4034, 1999;Jones et al., Blood 94:2871, 1999). Thus, the mAbs were cultured with B9cells in the presence of 100 ng/ml MBPvIL-6. As shown in FIG. 2, themAbs v6 m12.1.1, (ATTC Deposit No. PTA-2220, deposited Jul. 14, 2000),v6 m17.3.2 (ATTC Deposit No. PTA-2217, deposited Jul. 14, 2000), v6m27.1.2 (ATTC Deposit No. PTA-2218, deposited Jul. 14, 2000), and v6m31.2.4 (ATTC Deposit No. PTA-2219, deposited Jul. 14, 2000)demonstrated varying degrees of neutralizing activity against vIL-6(FIG. 2). The Ab v6 m31.2.4 exhibited most prominent neutralizingactivity in this bioassay. None of these mAbs suppressed theproliferation of B9 cells in response to hIL-6. The neutralizingactivity of anti-vIL-6 mAbs was further confirmed using the SKW6.4 humanB cells that are known to produce IgM in response to hIL-6 (Peppard etal., J Biol. Chem. 271:7281, 1996). Like hIL-6, MBPvIL-6dose-dependently stimulated IgM secretion in SKW6.4 cells (FIG. 3A).Maximal production of IgM was obtained with 2 μg/ml of MBPvIL-6 and 5–10ng/ml of hIL-6. vIL-6-induced IgM production by SKW6.4 cells wassuppressed by mAbs v6 m12.1.1, v6 m17.3.2, v6 m27.1.2 and v6 m31.2.4 butonly minimally by mAbs v6 m13.1.5 or v6m24.2.5 (FIG. 3B). The results ofthese vIL-6 bioassays indicate that clones v6 m12.1.1, v6 m17.3.2, v6m27.1.2 and v6 m31.2.4 have specific neutralizing activity againstvIL-6.

Other antibodies, such as polyclonal antibodies that specifically bindthe vIL-6 C-terminus (Science 274:1739, 1996) did not inhibit B9 cellproliferation. To demonstrate that not all antibodies that bind vIL-6are neutralizing, COS7 cells were transfected with expression vectorpMET7 containing vIL-6 gene or with expression vector pMET7 containingr6-LIv (a reverse control) gene. Serial dilutions of supernatants wereincubated in the presence or absence of anti-vIL-6 antiserum generatedin rabbits immunized with synthetic peptides of vIL-6 C-terminus(Science 274:1739,1996) with B9 cells (3×10³ cells per well) in a96-well plate at 37° C. for 72 h, including a 6-h terminal pulse with 1μCi/well of [³H]-thymidine. [³H]-thymidine incorporation was determinedafter cell harvesting onto glass fiber filters (see Table 2, below).

TABLE 2 Lack of neutralizing activity of a rabbit polyclonal antibodythat binds vIL-6 C-terminal amino acids THYSPPKFDR and PDVTPDVHDR¹Control vIL-6 containing supernatant supernatant plus (r60Llv) plusDilution of anti-vIL6 Ab anti-vIL6 Ab supernatants +² − +² − 1:2 13,653 9,057 3,002 2,853 1:4 8,532 7,247 1,787 1,195 1:8 3,681 3,344 1,3521,746 The results represent mean cpm of triplicate cultures;representative experiments of 5 performed: ¹Polyclonal antibodies werefrom Dr. Yuan Chang at Columbia University. These antibodies aregenerated against synthetic peptides [THYSPPKFDR and PDVTPDVHDR], andare described in U.S. Pat. No. 5,861,500 and in Science 274: 1739, 1996.²Rabbit antiserum against synthetic peptides from vIL-6 amino acidsequences was added at 1:250 dilution.

Thus, previous antibodies known to bind vIL-6 are not neutralizingantibodies.

Example 5 Mapping the Epitopes Recognized by the Neutralizing mAbs

The antigenic epitopes recognized by v6 m12.1.1, v6 m17.3.2, v6 m27.1.2and v6 m31.2.4 were mapped by ELISA using a panel of MBPvIL-6 fusionproteins (FIG. 4). Plate coating efficiency with fusion proteins wasconfirmed by reacting with polyclonal anti-MBPvIL-6 Ab. All six Abssimilarly recognized full-length vIL-6. The mAb v6 m13.1.5 bound to a 14amino acid stretch at the N-terminus of vIL-6, and the mAb v6 m24.2.5recognized 5 amino acids at the C-terminus of vIL-6 (FIG. 5). The mAbsv6 m12.1.1, v6 m17.3.2, v6 m27.1.2 and v6 m31.2.4 bound to full lengthMBPvIL-6 and to fragment M3 of MBPvIL-6. This fragment M3, but not othermutants, encompasses an internal 13 amino acids of the vIL-6 fragment atpositions 81–93, suggesting that these mAbs map to the vIL-6 fragmentinclusive of Asp⁸¹–Cys⁹³. The hIL-6 fragment corresponding to thisportion of vIL-6 constitutes the C-terminal part of the AB loop and thebeginning of helix B, which are included in the site 1 hIL-6 binding tothe IL-6Rα.

Example 6 A Cell-Free System to Determine the Interference of vIL-6Binding to sgp130

KSHV vIL-6 is one of the viral proteins that is implicated in thepathogenesis of AIDS-related malignancies. Although vIL-6 shows multiplebiological functions similar to those of cellular IL-6, the usage ofIL-6Rs by vIL-6 is still unclear. Cellular IL-6 exerts its actionsthrough a receptor complex consisting of a specific IL-6-bindingprotein, IL-6Rα, and a signal-transducing subunit, gp130. TheIL-6/IL-6Rα complex induces the homodimerization of two gp130 moleculesleading to a number of intracellular signaling events, includingactivation of the transcription factor NF-IL-6 via activation of theJAK/STAT signaling pathway (Hallek et al., J Virol. 73:5149, 1999; Tagaand Kisimoto, supra). Cellular IL-6 is unable to transduce signals inthe absence of IL-6Rα. Previously, the participation of IL-6Rα in vIL-6binding cells was deduced from experiments using the IL-6-dependentmurine B9 cells (Nicholas et al., Nat Med. 3:287, 1997). Supportiveevidence for a role of IL-6Rα in vIL-6 function derived from experimentsin which neutralization of IL-6Rα abrogated vIL-6-induced HIV-1 p24production in a human monocytic cell line (Gage et al., AIDS 13:1851,1999). However, other experiments showed that, whereas a combination ofanti-IL-6Rα and anti-gp130Abs blocked the proliferative effect of vIL-6in the human myeloma INA-6 cell line, anti-IL-6Rα Ab alone did not(Burger et al., Blood 91:1858, 1998). In addition, anti-IL-6Rα Abeffectively neutralized the response of HepG2 human hepatoma cells tohIL-6 but failed to block STAT activation by vIL-6 (Molden et al., JBiol Chem. 272:19625, 1997). By contrast, anti-gp130 Ab abrogated HepG2cell signaling in response to both hIL-6 and vIL-6. Moreover, BAF-130cells, murine pro-B cells expressing transfected human gp130 but notIL-6Rα exhibited a strong response to vIL-6 but not to hIL-6 (Molden etal., J Biol. Chem. 272:19625, 1997; Mulberg et al., J Immunol. 164:4672,2000). Thus, there seems to be a consensus that gp130 is used for vIL-6signaling, but the usage of IL-6Rα is subject to controversy.

A cell free system was created in order to examine the ability of themonoclonal antibodies to neutralize vIL-6. This system assessed thebiological activity of vIL-6/gp130 binding, described above.

The binding of vIL-6 to gp130 in a cell-free system could be inhibitedby neutralizing mAbs to vIL-6. To this end, vIL-6 (50 ng/ml) was firstincubated with control mouse IgG1 (20 μg/ml) or anti-vIL-6 mAbs (20μg/ml) overnight at 4° C., and then tested for binding to sgp130 boundto ELISA plates. Compared with the isotype control murine IgG1, the fouranti-vIL-6 neutralizing mAbs interfered with the binding of vIL-6 tosgp130, whereas the two non-neutralizing mAbs did not (FIG. 5). Theseresults strongly support the notion that vIL-6 can exert its bioactivitythrough direct binding to gp130.

Thus, evidence is provided herein that vIL-6 can bind to sgp130 in theabsence of sIL-6Rα in a cell-free system. Neutralizing mAbs directedagainst vIL-6 interfered with vIL-6 binding to sgp130. These findingsstrongly support the notion that vIL-6 exerts its biological functionsthrough direct binding to gp130.

The distinct biological activities of the gp130 family of cytokines havebeen explained by the occurrence of specific receptor chains. Unlike allother members of the IL-6 family, OSM shows a low-affinity bindingcapacity for gp130, which forms a high-affinity receptor complex withLIFR or OSMR (Tanaka et al., Blood 93:804, 1999). OSM transmits agrowth-suppressing signal in NIH3T3 cells through a high affinityreceptor composed of gp130 and OSMR (Ichihara et al., Blood. 90:165,1997). Although NIH3T3 cells do not express the IL-6Rα and thus do notnormally respond to IL-6, IL-6 combined with sIL-6Rαcan suppress thegrowth of NIH3T3 cells (Ichihara et al., Blood 90:165, 1997). Bycontrast, vIL-6 can stimulate NIH3T3 cells to produce vascularendothelial growth factor in the absence of sIL-6Rα (Robledo et al.,Cytokine 9:666, 1997; Saijonmaa et al., Am J Physiol. 275:H662–37, 1998;Bernard et al., Circ Res. 85:1124, 1999). These observations havesuggested that vIL-6 may exert its biological activities in a fashionsimilar to OSM. Importantly, OSM is a potent inducer of IL-6 in certaincell types (Wan et al., J Virol. 73:8268, 1999; Gage et al., AIDS13:1851, 1999; Kellam et al., J Virol. 73:5149, 1999). IL-6 geneexpression is regulated by various cis-acting elements, depending uponcell type and nature of the activating agent (Kishimoto et al., Blood.86:1243, 1995). Should vIL-6 be able to induce cells to secreteendogeous IL-6, like OSM, a potential explanation for the contradictoryobservations about vIL-6 receptor usage would be possible.

Thus, six clones of mAbs were produced against vIL-6. Although the wholemolecule of vIL-6 was used for immunization, none of six Abs recognizescellular IL-6. Of note, even a polyclonal Ab raised in rabbits againstvIL-6 failed to recognize hIL-6 and mIL-6. Further, polyclonalanti-mIL-6 Ab that cross-react to hIL-6 did not detect vIL-6.

KSHV vIL-6 shares extensive functional similarity with hIL-6 and mIL-6although vIL-6 requires up to 1,000-fold larger amounts of protein inbioassays using IL-6 dependent cell lines. Structural differencesbetween vIL-6 and cellular IL-6 and differences in receptor bindingaffinity may account for the greater amounts of vIL-6 being required forbiological activities in vitro.

In the case of vIL-6, it is possible that this viral cytokine can bindto a specific receptor yet to be defined capable of forming functionalheterodimers with gp130. Thus, vIL-6, a viral cytokine homologue of acellular cytokine, appears to share, at least in part, the receptorsutilized by the cellular cytokine.

vIL-6 was originally identified in KSHV-positive PEL cells (Moore etal., Science. 274:1739, 1996). This viral protein can stimulate thegrowth of PEL cells in an autocrine/paracrine fashion (Jones et al.,Blood 94:2871, 1999) and induce vascular endothelial growth factor,which is critical for the growth of PEL and KS in vivo (Aoki et al.,Blood. 93:4034, 39–43, 1999). Abundant vIL-6 expression has also beendetected in the mantle zones of multicentric Castleman's disease(Parravicini et al., Am J Pathol. 151:1517, 1997). vIL-6-positive cellsin Castleman's disease were negative for CD3, CD20, CD30, CD138, CD45RO,CD68 and EMA. By contrast, hIL-6 is expressed mainly in the germinalcenters of Castleman's disease (Yoshizaki et al., Blood 74:1360, 1989).Although IL-6Rα is not expressed on resting B cells, vIL-6 couldstimulate those cells via direct binding to gp130.

The absence of IL-6Rα expression was previously demonstrated in AIDS-KScells (Murakami-Mori et al., J Clin Invest. 96:1319, 1995), whereas highlevels of gp130 expression were noted. OSM, but not IL-6 or LIF,stimulated the growth of AIDS-KS cells, and anti-gp130 Ab completelyabolished OSM-induced growth stimulation of AIDS-KS cells (Murakami-Moriet al., J Clin Invest. 96:1319, 1995). A proportion of HIV-positivepatients have detectable vIL-6 in sera and body fluids (Aoki et al.,Blood. 96:1599, 2000; Aoki et al., Blood 97:2173, 2001; Aoki et al.,Blood. 97:2526, 2001), which may promote the development ofAIDS-associated malignancies by constitutive phosphorylation of gp130 orby inducing vascular endothelial growth factor. Deregulation of cellularIL-6 expression is known to contribute to tumor development, suggestingthat KSHV-derived vIL-6 could be part of a viral strategy to promotemalignant transformation. Neutralizing activity of anti-vIL-6 Abs mayprovide a new experimental therapeutic for KSHV-associated disorders.

Example 7 Gp130 Binding Surfaces are not Fully Shared by Human and vIL-6

It was examined if the vIL-6 epitope recognized by the neutralizing mAbsis used as a direct binding surface for gp130. Murine IL-6 isspecies-specific whereas human IL-6 binds to both human and murinereceptors. To exclude a potential contribution of vIL-6-induced cellularIL-6, murine BAF-B03 cells stably transfected with human gp130 wereutilized. This cell line is known to respond to vIL-6 but not to humanIL-6 in the absence of sIL-6Rα (Molden et al., J Biol Chem 1997;272:19625–19631; Narazaki et al., Blood 1993; 82:1120–1126). Asexpected, human IL-6/sIL-6Rα induced DNA synthesis in BAF-130 cells. Inagreement with its lower binding affinity, vIL-6 had much lower specificactivity than human IL-6/sIL-6Rα. The vIL-6 deletion mutant MBPvIL-6 M3,which contains the region of Asp⁸¹-Cys⁹³ within site I but lacks fullsite II and III comprising the reported vIL-6 interface with gp130(FIGS. 9 and 11), did not induce DNA synthesis in BAF-130 cells orcompete for vIL-6 or human IL-6/sIL-6Rα binding to gp130. This suggestedeither that this vIL-6 epitope is not part of the vIL-6 binding surfaceto gp130, as shown by the vIL-6 crystal structure, or that this fragmentalone is not sufficient for biological activity. Neither parentalBAF-B03 cells nor mock transfectants responded to vIL-6.

It was then examined if human and vIL-6 share the same gp130 bindingsites using a panel of anti-gp130 mAbs. These Abs are known to recognizespecific functional sites of gp130 for signal transduction by IL-6-typecytokines (Wijdenes et al., Eur J Immunol 1995; 25:3474–3481;Muller-Newen et al., J Biol Chem 2000; 275:4579–4586). As reportedpreviously, all three anti-gp130 mAbs B-R3, B-P4 and B-P8, but not anisotype control, inhibited human IL-6-induced DNA synthesis in BAF-130cells. By contrast, when stimulated with vIL-6, mAbs B-R3 and B-P4, butnot B-P8, suppressed DNA synthesis in BAF-130 cells (Muller-Newen etal., J Biol Chem 2000; 275:4579–4586; Kurth et al., J Immunol 2000;164:273–282). The mAb B-P4 recognizes the membrane-proximalextracellular part of gp130 (D4D5D6) that is critical forhomodimerization of gp130 (Kurth et al., J Immunol 2000; 164:273–282).The B-R3 mAb interferes with gp130 binding by all IL-6-family cytokines,whereas B-P8 mAb interferes with gp130 binding by human IL-6 and ciliaryneurotrophic factor (Wijdenes et al., Eur J Immunol 1995; 25:3474–3481;Kurth et al., J Immunol 2000; 164:273–282). The mAbs B-R3 and B-P8recognize the epitopes in gp130 D2 and D3, respectively, whichconstitute the cytokine binding module (Wijdenes et al., Eur J Immunol1995; 25:3474–3481; Muller-Newen et al., J Biol Chem 2000;275:4579–4586). These results provide evidence that human and vIL-6share an epitope in D2 of gp130, and that both cytokines inducehomodimerization of gp130. In addition, the result show that vIL-6 doesnot bind to the epitope in gp130 D3 which is critical to human IL-6function. vIL-6 shows biological functions similar to those of cellularIL-6, however, the interaction between vIL-6 and its receptor(s) appearsto be different from that of cellular IL-6. Evidence is provided hereinthat vIL-6 does not bind to IL-6Rα in a cell-free system. By bothsurface plasmon resonance and/or ELISA, vIL-6 bound gp130 without a needfor IL-6Rα and failed to bind IL-6Rα. In addition, mAb v6 m24.2.5recognizing the C-terminal end of helix-D of vIL-6 did not showneutralizing activity in bioassays. In contrast to these results withvIL-6, the corresponding C-terminal end of helix-D in human IL-6 isincluded in the binding face to IL-6Rα, and the binding of mAb to thisregion totally abrogated human IL-6 activity by blocking the ligandinteraction to IL-6Rα (Brakenhoff et al., J Immunol 1990; 145:561–568).

Using the structure of the growth hormone/growth hormone receptorcomplex as a paradigm for cytokine receptor complex assembly, IL-6-typecytokines are believed to have three topologically discrete sites ofinteractions with their receptors (FIG. 9). Site I, if used, is alwaysengaged by a non-signaling receptor: IL-6Rα, IL-11Rα or ciliaryneurotrophic factor receptor-α. Site II is always engaged by gp130, andsite III by a second signaling receptor gp130, OSMR or LIFR (Bravo etal., EMBO J. 2000; 19:2399–2411). Within gp130, three binding epitopeshave been identified as critical to its activation by human IL-6/IL-6Rα:one epitope involves the Ig-like domain (D1); another epitope is locatedin the cytokine binding module (D2D3); and the other is located in themembrane-proximal extracellular domains (D4D5D6) (Kurth et al., JImmunol 2000; 164:273–282). According to the crystallographic analysisof the vIL-6sgp130(D1D2D3) complex (Chow et al., Science 2001;291:2150–2155), sequence alignment of human and vIL-6 shows that contactresidues seen in the structure of the vIL-6/gp130 complex are in thesame positions as human IL-6-gp130 contact residues previously mapped bymutagenesis (Simpson et al., Protein Sci 1997; 6:929–955). In cellculture, the membrane-proximal extracellular part of gp130 (D4D5D6) iscritical for vIL-6 mediated-signaling, even though vIL-6 can form atetrameric complex with gp130 (D1D2D3) (Chow et al., Science 2001;291:2150–2155). Further, although human and vIL-6 share an epitope ingp130 D2, vIL-6 does not appear to utilize an epitope in gp130 D3 thatis critical to human IL-6 function. Together with the evidence thatvIL-6 does not bind IL-6Rα, these results are consistent with theprevious observation that vIL-6 did not compete with the human IL-6superantagonist Sant7 for human myeloma cell stimulation.

In the studies described herein, the neutralizing mAbs directed againstvIL-6 recognize a region of the C-terminal part of the AB-loop and thebeginning of helix-B. The binding of mAb to this region can change themobility of helix-B and interfere with the ability of site II and III toachieve a proper orientation. Without being bound by theory, the bindingof human IL-6 to IL-6Rα could modify a loop-helix interaction intoactive conformation before binding to gp130. In the case of vIL-6, theabsence of a specific receptor chain may result in the low bindingaffinity of vIL-6 to gp130 by failure to be locked in a high affinityconformation.

Example 8 Use of a vIL-6 Specific Binding Agent to Diagnose PEL:Materials and Methods

Primary effusion lymphoma (PEL) is a peculiar and infrequent type ofnon-Hodgkin's lymphoma that arises predominantly in humanimmunodeficiency virus (HIV)-infected individuals. PEL displays liquidgrowth in the serous cavities of the body, often in the absence of aclearly identifiable tumor mass (Cesarman et al., Semin Cancer Biol9:165–174, 1999). In most cases, PEL cells are dually infected withEpstein-Barr virus and Kaposi's sarcoma-associated herpesvirus (KSHV;also known as human herpesvirus 8) (Nador et al. Blood 88:645–656, 1996;Gaidano et al., Blood 90:4894–4900, 1997), and produce severalcytokines, including a viral homologue of interleukin (IL)-6 (vIL-6)(Moore et al., Science 274:1739–1744, 1996; Nicholas et al., Nat Med3:287–292, 1997; Neipel et al, J Virol 71:839–842, 1997).

Although HIV-associated lymphomagenesis is poorly understood,experiments in vitro and transgenic models have shown that HIV-derivedproteins can activate a number of cellular genes. For example, the HIVtransactivator protein Tat has been reported to promote expression ofhIL-6 and hIL-10 in lymphoid cells (Kundu et al., Blood 94:275–282,1999). Further, HIV infection and soluble factors released fromHIV-infected cells can induce lytic replication of KSHV in a PEL cellline (Varthakavi et al., J Virol 73:10329–10338, 1999). hIL-6expression, which is constitutive in PEL cell lines, is downregulatedduring KSHV lytic replication induced by phobor esters (Asou et al.,Blood 91:2475–2481, 1998). By contrast, vIL-6 expression, which is lowin PEL cell lines latently infected with KSHV, is markedly inducedduring KSHV lytic replication (Sarid et al., J. Virol. 72(2):1005–1012,1998). The potential relationships between HIV load and the induction ofselected cellular and KSHV-derived cytokines was investigated.

In order to evaluate the role of vIL-6 in PEL, the following materialsand methods were used:

Patients: Body cavity effusions from 8 AIDS patients with PEL, 21HIV-negative patients with malignancies or congestive heart failure, and4 AIDS patients with inflammatory processes without PEL were obtainedfrom the AIDS Malignancy Bank (National Cancer Institute, Bethesda, Md.)and from our Institutes. The diagnosis of PEL was based on clinicalpresentation, histology of the effusion cells, and the presence of KSHVin the lymphoma cells. HIV-RNA load was measured by standard techniques.

Enzyme-Linked Immunosorbent Assays (ELISA) for vIL-6, hIL-6 and hIL-10:The ELISA for vIL-6 utilized a mouse monoclonal and rabbit polyclonalantibodies raised against recombinant vIL-6 (Jones et al., Blood94:2871–2879, 1999; Aoki et al., Blood 93:4034–4043, 1999; Aoki et al.,Blood 94:431 a, 1999). Polystyrene plates (Immunol 1B; DynexTechnologies, Chantilly, Va.) were coated with mouse monoclonalanti-vIL-6 antibody (v6 m12.1.1; 4 μg/ml in carbonate buffer, pH 9)overnight at 4° C. After washing the plates with PBS containing 0.05%Tween 20 (PBS-T) and blocking with SuperBlock (Pierce, Rockford, Ill.),test samples were added in triplicate to the wells (initial dilution1:10 in PBS-T). Plates were incubated overnight at room temperature, andwashed with PBS-T. Rabbit polyclonal anti-vIL-6 antibody (0.5 μg/ml) wasadded to the wells in PBS-T containing 0.5% bovine serum albumin(PBS-T/BSA). Plates were incubated for 2 h at room temperature andwashed. Affinity-purified, human serum protein absorbed goat anti-rabbitIgG antibody conjugated to alkaline phosphatase (Sigma, St. Louis, Mo.;1:400 dilution in PBS-T/BSA) was added. Plates were incubated at roomtemperature for 1.5 hours, washed with PBS-T, p-nitrophenolphosphatesubstrate (Sigma) added to the wells, and plates were read at 405 nmwith λ correction at 595 nm. A purified fusion protein ofmaltose-binding protein (MBP: 42.7 Kd) and amino acids 22–204 of vIL-6(21.6 Kd) was used as the standard (Aoki et al., Blood 93:4034–4043,1999). The concentration of vIL-6 was calculated from absorbance valuesin relation to the standard curve, correcting for the presence of theMBP fusion protein in the vIL-6 standard (vIL-6 corresponds to 33.6% ofMBPvIL-6). hIL-6 and hIL-10 were measured by commercially availableELISA kits (R & D Systems, Minneapolis, Minn.).

Western Blotting: Western blotting for vIL-6 and hIL-6 was performed asdescribed previously (Aoki et al., Blood 93:4034–4043, 1999). Rabbitpolyclonal and a mouse monoclonal (v6 m12.1.1, see above) antibodiesagainst vIL-6, and mouse monoclonal antibody against hIL-6 (MAb206; R&DSystems) were used as the primary antibodies. MBPvIL-6 was cleaved withfactor Xa (New England BioLabs, Beverly, Mass.).

Statistical Analysis: The non-parametric Spearman-Rho test was used tomeasure the significance of correlations between groups.

Example 9 Use of a vIL-6 Specific Binding Agent to Detect PEL: Results

vIL-6 exhibits 24.7% amino acid identity to hIL-6 (Moore et al., Science274:1739–1744, 1996). Thus, an assay for vIL-6 must be able todistinguish it from hIL-6. As assessed by both immunoblotting and directELISA, neither a rabbit polyclonal nor a mouse monoclonal antibodiesraised against recombinant vIL-6 recognize hIL-6. Using theseantibodies, a vIL-6 ELISA was developed. This assay displays a lowerlimit of sensitivity calculated at approximately 30 pg/mL of vIL-6, andis linear between 30 and 3,360 pg/mL of vIL-6. This ELISA does notdetect hIL-6 (FIG. 6A), and a commercial hIL-6 ELISA kit (R&D Systems)does not detect vIL-6 (FIG. 6B).

PEL cell cultures consist largely of cells latently infected with KSHV,with a minority of cells undergoing lytic KSHV replication (Renne etal., Nat Med 2:342–6, 1996). Treatment with the phorbol ester12-O-tetradecanoylphorbol-13-acetate (TPA) rapidly induces lytic KSHVreplication. The KSHV-positive PEL cell lines BC-1, BCP-1, and BCBL-1were found to release vIL-6 into the supernatant, as did the vIL-6transfected NIH3T3 v60 cells (Table 3) (Aoki et al., Blood 93:4034–4043,1999).

TABLE 3 Detection of vIL-6 in the culture supernatants of cell lines¹culture without TPA culture with TPA cell line (vIL-6 pg/mL) (vIL-6pg/mL) BC-1 ^(2, 3) 2880 26300 BCP-1 ² 19200 118100 BCBL-1 ² 1680 29100Daudi ³ <30 <30 v6O ⁴ 88600 ND VDS-O1 ^(3, 5) <30 ND ¹ To prepareconditioned media, suspension cells were seeded in 12-well plates at 1 ×10⁶ cells/well in 2.5 ml of RPMI1640 medium supplemented with 10% fetalbovine serum and cultured with or without 20 ng/mL TPA (Sigma) for 48hours. Adherent cells were seeded in 6-well plates at 1 × 10⁶ cells/wellin 2.5 ml culture media, and cultured for 48 hours. The concentration offetal bovine serum in each supernatant was adjusted to be 10%.Datarepresent the mean of duplicate assays. ² KSHV-positive cell lines(Moore et al.,. Science 274:1739–1744, 1996; Renne et al., Nat Med2:342-6, 1996; Cesarman et al., Blood 86:2708–2714, 1995). ³EBV-positive cell lines (Aoki et al., Blood 94:4247–4254, 1999). ⁴NIH3T3 cells stably transfected with vIL-6-expression vector (Aoki etal., Blood 93:4034–4043, 1999). ⁵ Lymphoblastoid cells stablytransfected with hIL-6-expression vector. (Tanner et al., J Clin Invest88:239–247, 1991). ND not determined.

Addition of TPA to PEL cells enhanced vIL-6 release in the culturesupernatants. No vIL-6 was detected in the supernatants from theKSHV-negative Burkitt's lymphoma cell line Daudi or the EBV-immortalizedVDS-O1 cell line which is transfected with a hIL-6 expression vector(Tanner et al., J Clin Invest 88:239–247, 1991).

vIL-6 was detected in 6 of 8 PEL effusions (mean 13,884 pg/mL), but wasundetectable in the 21 control benign or malignant effusions fromHIV-negative individuals (not shown; p<0.0001, Fisher's Exact test).vIL-6 was also undetectable in 4 non-malignant effusions from patientswith AIDS (not shown). HIV RNA was examined in 8 malignant lymphomatouseffusions (Table 4).

TABLE 4 HIV load and cytokines in AIDS-PEL effusions HIV RNA casecopies/mL¹ vIL-6 (pg/mL)² hIL-6 (pg/mL)³ hIL-10 (pg/mL)³ 1 59796 666306787 87222 2 inhibitory⁴ 9670 15561 231454 3 >750000 <300 957 2521297 48741 <300 37494 <8 5 66353 1390 6856 66 6 111920 16350 4935 103414 72169 10120 8395 8327 8 88801 6913 15097 1342318 ¹HIV-RNA was measured byquantitative RT-PCR kit (Roche Amplicor HIV-test). ²Due to the initial1:10 sample dilution, the lower limit of ELISA sensitivity was set at300 pg/mL of vIL-6. ³Data represent the mean of triplicate assays. ⁴Testresults indicated the presence of an inhibitor of RT-PCR in this sample.

Except for one sample where test results could not be evaluated becauseof an inhibitor, HIV RNA was detected in all PEL effusions (mean 562967copies/mL). hIL-6 was detected in all AIDS-PEL effusions (mean 12010pg/mL) and in all control HIV-negative effusions (mean 41,737 pg/mL;range 127–624,870 pg/mL). No significant correlation was observedbetween vIL-6 and hIL-6 levels in PEL effusion (r=−0.2275; p=0.5878).hIL-10 was detected in 7 of 8 PEL effusions (mean 536,762 pg/mL). Astatistically significant association was observed between HIV load andhIL-10 levels (r=0.7857; p=0.0362). However, no significant associationwas noted between HIV load and vIL-6 (r=−0.1622; p=0.7283) and hIL-6levels (r=−0.6786; p=0.0938).

Thus, PEL effusions generally contain vIL-6, hIL-6, hIL-10 and HIV RNA.These PEL effusions contain high levels of VEGF (mean 3,977 pg/mL; range1,133–11,417 pg/ml) (Aoki et al., Blood 95:1109–1110, 2000). PEL cellsare a likely source of vIL-6, hIL-6, hIL-10 and VEGF detected in thelymphomatous effusions because PEL cell lines can express all theseproteins in culture (Jones et al., Blood 94:2871–2879, 1999; Aoki etal., Blood 94:4247–4254. 1999). VEGF, which stimulates vascularpermeability and may facilitate the accumulation of PEL effusions invivo, was required for a PEL cell line to form effusion lymphomas inmice (Aoki et al., Blood 94:4247–4254. 1999). hIL-6 and vIL-6,individually, can stimulate the expression of VEGF in tissues. In vitro,hIL-10 and vIL-6 serve as autocrine growth factors for PEL cell lines(Jones et al., Blood 94:2871–2879, 1999), and may promote PEL cellgrowth in the body cavities. The observation that PEL effusionsgenerally contain vIL-6 which can stimulate PEL cell growth and promotethe accumulation of effusions further suggests that this vIL-6 plays acritical role in PEL pathogenesis.

Example 10 Detection of vIL-6 in Castleman's Disease: vIL-6 LevelsReflect Disease Activity and Parallel Effective Treatment of the Disease

A 40-year-old HIV-positive homosexual man presented with a sore throat,non-productive cough, night sweats, fever, diarrhea, marked fatigue andweight loss. The patient had received highly active antiretroviraltherapy (HAART) for three years, including lamivudine (3TC: 150 mg twiceper day), stavudine (d4T: 40 mg twice per day) and nelfinavir (NFV: 2250mg per day). HIV load had been stable at less than 1,200 copies/mL since1996. CD4⁺ T-cell counts had remained over 300 cells/mm (Aoki et al.,manuscript submitted). On physical examination, the patient hadlymphadenopathy, abdominal distention with hepatosplenomegaly, abdominaltenderness and skin rash but no evidence of Kaposi's sarcoma. Oraladministration of prednisone (30 mg of per day) was started to relievesystemic inflammatory symptoms. A cervical lymph node biopsy displayedabnormal histology that shows the typical features of mixed plasmacell/hyaline vascular type of multicentric Castleman's disease(Oksenhendler et al., AIDS 10:61–7, 1996). The lymph node displayedconcentric layers of small lymphocytes surrounding the germinal centersand plasma cells infiltration in the interfollicular areas. Due to adramatic improvement of systemic symptoms, prednisone was tapered after10 days, and administration of Foscarnet (7 g twice per day), ananti-herpesvirus agent, started. The patient was splenectomized inSeptember, 1999, and has been well for nine months. No serum sampleshave been available for analysis since August, 1999.

The serum samples were analyzed retrospectively. vIL-6 was measured by avIL-6-specific (does not recognize hIL-6) ELISA established in ourlaboratory (see above). hIL-6 was measured by a commercially availableELISA kit (R&D Systems, Minneapolis, Minn.) which does not detect vIL-6.

The presence of the KSHV antigens was examined in tissues byimmunohistochemistry using monoclonal antibodies againstlatency-associated nuclear antigen LANA (Dupin et al., Proc Natl AcadSci USA 96:4546–51, 1999) and vIL-6 (as described herein), according topreviously described methods (e.g. see Aoki et al., Blood. 93:4034–4043,1999). Immunohistochemical staining of the lymph node for hIL-6 wasperformed using monoclonal antibodies against hIL-6 (MAB206; R & Dsystems). HIV-RNA load was measured by standard techniques.

KSHV infection in the patient was confirmed by the immunochemicaldetection of the KSHV nuclear antigen LANA in a lymph node biopsyspecimen. A vIL-6 specific monoclonal antibody detected expression ofvIL-6 in the same lymph node specimen by immunohistochemistry. TheKSHV-LANA positive and vIL-6 positive cells localized predominantly tothe mantle zone of the lymph node with similar proportion. By contrast,expression of hIL-6, which has previously been detected in the germinalcenters of certain Castleman's disease cases, was not detectable byimmunohistochemistry in this lymph node. The vIL-6, hIL-6 and HIV-RNAload in the patient's serum was measured at various time intervals,beginning 10 days after initiation of steroid treatment (FIG. 7). SerumvIL-6 was initially detected at 4,756 pg/mL. However, vIL-6 decreased toundetectable (less than 300 pg/mL) levels over the next 10 days andsubsequently remained undetectable. The HIV RNA load also presentedremarkable changes during this period. For approximately 3 years priorto the onset of Castleman's disease, the HIV RNA load in this patienthad remained at less than 400 copies/mL. No serum samples were availablefrom these years for vIL-6 measurement. After Castleman's disease wasdiagnosed, the HIV RNA load peaked at 146,460 copies/m, followed by arapid decrease to 792 copies/mL on day 21 of treatment (FIG. 7). Ofnote, the patient continued to receive the same antiretroviral drugregimen he had received during the previous 3 years. Thus, vIL-6 and HIVRNA serum levels displayed parallel decreases over a 20 day period ofobservation following initiation of steroid treatment for multicentricCastleman's disease (r=0.882: p=0.0053, Spearman Rho test). By contrast,serum levels of hIL-6 fluctuated at low levels (range <1.0–10.8 pg/mL)throughout this period. This demonstrates that decreased levels ofcirculating vIL-6 reflect an effective treatment of Castleman's disease.Thus, circulating vIL-6 levels can be used to monitor Castleman'sdisease activity.

Deregulation of hIL-6 has been previously implicated in the pathogenesisof Castleman's disease. The germinal centers of hyperplastic lymph nodesin multicentric Castleman's disease were reported to express abundanthIL-6, and serum levels of hIL-6 were found to be abnormally elevated(Parravicini et al., Am J Pathol. 151:1517–22, 1997; Beck et al., N EnglJ Med. 330:602–5, 1994). In selected patients with Castleman's disease,neutralizing antibodies against human (non-viral) IL-6 may have exerteda therapeutic effect (Beck et al., op. cit.). In contrast to hIL-6, therole of KSHV-encoded vIL-6 in Castleman's disease is unclear. Virtuallyall HIV-positive cases of multicentric Castleman's disease and nearly50% of HIV-negative cases are infected with KSHV (e.g. see Soulier etal. Blood 86:1276–80, 1995) and all KSHV-positive Castleman's diseasetissues were found to express vIL-6 (Parravicini, supra). Studies inmice suggested that vIL-6 may directly stimulate B cell proliferationand differentiation suggesting its potential role in the pathogenesis ofmulticentric Castleman's disease (Nicholas et al., Nat Med. 3:287–92,1997). In addition, since vIL-6 can induce vascular endothelial growthfactor, a potent angiogenic factor, vIL-6 could indirectly contribute toincreased lymphatic angiogenesis that is often noted in Castleman'sdisease lesions. The results described herein demonstrated that vIL-6was detected in the circulation of an AIDS patient with multicentricCastleman's disease, but became undetectable soon after initiation ofsteroid treatment that resulted in clinical remission.

Recent studies have focused on the relationship between HIV and KSHVinfection. Some experiments in vitro have shown that vIL-6 can activateHIV-1 replication in human monocytes (Gage et al., AIDS 13:1851–5,1999), others that HIV infection and soluble factors released fromHIV-infected cells can induce lytic KSHV replication in a PEL cell line(Varthakavi et al., J. Virol. 73:10329–38, 1999). In the patientdescribed herein, the onset of multicentric Castleman's disease wasassociated with a marked increase of the HIV RNA load followed by arapid return to lower levels. The anti-retroviral therapy had notchanged in this patient so it is unlikely that either the increase orthe decrease in HIV-RNA load is attributable to this therapy. HIV-1 isnot known to infect B cells in Castleman's disease lesions, so that itis unlikely that tumor burden per se is responsible for the increase inHIV-RNA load. Rather, it is more likely that factors derived fromCastleman's disease lesions, including vIL-6, may have activated HIVreplication in this patient.

Initiation of steroid treatment was associated with a rapid antitumorresponse, a decrease in the patient's HIV RNA load and a paralleldecrease of vIL-6 serum levels. This outcome is consistent with thenotion that the reduction of Castleman's disease burden induced bysteroids also reduced factors derived from these tissues, includingvIL-6. In turn, reduction of vIL-6, and perhaps other factors derivedfrom Castleman's disease tissues, may have removed a signal for HIVreplication, and allowed HIV RNA load to return to levels measured priorto the onset of Castleman's disease. While the factors contributing tothe onset of multicentric Castleman's disease are likely multiple, theremarkable association between circulating vIL-6 levels and Castleman'sdisease status shown here, combined with previous information on thebiological activities of this cytokine, supports a role of vIL-6 in thepathogenesis of this lymphoproliferative disorder. Administration of aspecific binding agent which neutralizes a biological activity of vIL-6can thus be utilized to treat Castleman's disease (see Example 11,below).

Example 11 Detection of Kaposi's Sarcoma-Associated Herpesvirus(KSHV)-Encoded Interleukin-6 in KSHV-Related Disorders

Methods

Patients and Blood Donors: Sera from healthy controls, HIV-positiveindividuals, and patients with KS, PEL or MCD were collected from bloodbanks and clinical centers located in the United States. Samples werestored at −70° C. prior to testing. RNA load of HIV was determined bystandard techniques. Counts for CD4 or CD8 positive cells weredetermined by flow cytometry.

ELISA for viral and human IL-6: An ELISA for vIL-6 was performed asdescribed herein. Briefly, polystyrene plates (Immunol 1B; DynexTechnologies, Chantilly, Va.) were coated with mouse monoclonalanti-vIL-6 antibody (v6 m12.1.1; 4 μg/mL in carbonate buffer, pH 9).After washing the plates with PBS containing 0.05% Tween 20 (PBS-T) andblocking with SuperBlock (Pierce, Rockford, Ill.), test samples wereadded in triplicate to the wells (initial dilution 1:10 in PBS-T).Plates were incubated overnight, washed with PBS-T, and rabbitpolyclonal anti-vIL-6 antibodies (0.5 μg/mL) were added to the wells inPBS-T containing 0.5% bovine serum albumin (PBS-T/BSA). Plates wereincubated for 2 h, washed, and affinity-purified, human serum proteinabsorbed goat anti-rabbit IgG antibody conjugated to alkalinephosphatase (Sigma, St. Lois, Mo.; 1:400 dilution in PBS-T/BSA) wasadded. Plates were incubated for 1.5 hours, washed, andp-nitrophenolphosphate substrate (Sigma) added to the wells. Theabsorbance values were read at 405 nm with λ correction at 595 μm. Apurified preparation of E. coli-derived recombinant vIL-6 was used asthe standard. The concentration of vIL-6 was calculated from absorbancevalues in relation to the standard curve. Quantification of human IL-6was performed using a human IL-6 Quantikine kit (R & D, Minneapolis,Minn.) that does not detect vIL-6.

Statistical Analysis. The Fisher's Exact test was used to examine therelationship between vIL-6 and sex, HIV risk factors, distribution ofKS, lymphadenopathy, use of anti-herpesvirus agents and use ofanti-retroviral agents. Mann-Whitney U test was used to examine therelation between seroprevalence of vIL-6 and HIV load, counts of CD4⁺ Tcells, CD8⁺ T cells, and platelets.

Results: As described above, a vIL-6-specific ELISA that does not detecthuman IL-6 has been developed. The lower limit of assay sensitivity wasdetermined to be 30 pg/ml of vIL-6. Serum samples were run at an initial1:10 dilution, and the lower limit of ELISA sensitivity for serum wasthus set at 300 pg/ml. vIL-6 was measurable in 1 of 40 blood donors(FIG. 8). To test for false positive ELISA reactions, we repeated theassays without coating the plates with capture monoclonal antibodyagainst vIL-6. Both previously positive samples were negative in theabsence of coating, providing evidence that positive reactions were notattributable to nonspecific binding of the detection antibodies orsubstrate.

The vIL-6 levels in serum from patients with AIDS-associated KS wasmeasured (FIG. 8). vIL-6 was detectable in serum from 16 of 34 patients(45.7%; range 370–7,460 pg/mL). To better evaluate the results of vIL-6testing in patients with AIDS KS, serum vIL-6 was measured in a group of30 HIV-positive individuals without KS, PEL or MCD. Most (90.6%) ofthese HIV-positive individuals were homosexual males who met thediagnostic criteria of AIDS. vIL-6 was detected in 19 of these 30HIV-positive individuals without evidence of KSHV-diseases relateddiseases (65.5%; range 301–15,060 pg/mL). Thus, the serum levels ofvIL-6 in the HIV-positive patients with KS and those without KS, PEL orMCD were similar (p=0.1846).

In 10 of the HIV-positive patients without KS, PEL or MCD, serum sampleswere obtained at more than one time. Thus, vIL-6 was measured in allavailable samples from these patients (Table 5).

TABLE 5 Clinical events and laboratory findings in 10 patients with HIVinfection Sampling vIL-6 hIL-6 HIV RNA CD4/CD8 antiretroviral therapyanti-KS Case MM/YY KS (pg/mL) (pg/mL) copies/mL cells/μL NRTIs NNRTIsPIs treatment 1 12/87  + <300 <1.0  60231  19/477 AZT, ddC — — — 4/88 +370 60.7 not available  2/317 AZT — — Irradiation 2 9/93 + <300 3.8 <200 242/591 AZT — — — 4/99 + 5361 23.3 113846 122/514 ABV NVP IDV VCV,ADM 6/99 + 7460 <1.0 194258  32/255 ABV NVP IDV VCV, ADM 3 6/87 + <300<1.0  33776 875/410 AZT — — — 7/87 + <300 <1.0  30926 972/1458 AZT — — —4 5/89 + <300 4.6  8376 441/2001 AZT — — — 8/89 + 470 25.0  2771502/2279 — — — — 5 3/98 + 530 6.2  <200 601/1328 AZT, 3TC — NFVThalidomide 4/99 + 714 3.7  <200 714/1006 AZT, 3TC — NFV IL-12 6/99 +671 3.8  <200 505/1173 AZT, 3TC — NFV IL-12 6 8/98 + <300 65.9  <200275/1132 D4T, 3TC — IDV — 4/99 + <300 <1.0  <200 287/670 D4T, 3TC — IDVThalidomide 5/99 + <300 7.6  <200 345/782 D4T, 3TC — IDV ADM 7 4/99 +<300 <1.0  <200 144/883 D4T, 3TC — NFV — 6/99 + <300 3.8  <200 137/906D4T, 3TC — NFV ADM 8 4/99 + 4507 <1.0  10176 260/398 AZT, 3TC — NFV CDV6/99 + 4753 <1.0  9332 325/340 ABV NVP SQV, RTV ADM 9 4/99 − 10384 <1.0130240 441/2001 F-ddA, D4T — NFV — 6/99 − 11311 <1.0 114598 502/2279ddI, ABV — SQV, RTV — 10 4/99 − <300 <1.0 180795  28/936 F-ddA, D4T — —— 6/99 − <300 2.4 172227  32/1116 — — — — MM/YY: month/year, ABV:abacavir, 3TC: lamivudine; D4T: stavudine; NFV: nelfinavir; SQV:saquinavir; IDV: indinavir; RTV: ritonavir; CDV: cidofovir; VCV:valacyclovir; NVP: nevirapine; ADM: adriamycin

The serum levels of human IL-6 were also measured, and information wascollected on multiple disease-related factors such as HIV-RNA load, CD4+and CD8+ cell counts, and treatment for HIV and herpes virus (Table 4).Serum levels of vIL-6 levels generally remained stable over a period ofmonths as did the HIV RNA load on anti-retroviral treatment, whereas CD4cell counts fluctuated. Human IL-6 was either undetectable or detectedat low levels (<1.0–116 pg/mL; median 2.4 pg/mL) in all sera. In thissmall group, no clear association was noted between serum levels ofvIL-6 and HIV-RNA load (Table 5).

The analysis was therefore extended to ascertain potential associationsbetween serum levels of vIL-6 and factors relevant to HIV disease to theentire HIV-positive group where serum vIL-6 had been measured. Thisanalysis (Table 6) included all 64 HIV-positive patients with (n=34) orwithout (n=30) KS, where serum vIL-6 was either detected (n=31) or notdetected (n=33).

TABLE 6 Characteristics of HIV-positive patients by vIL-6 levels.^(a)vIL-6 negative vIL-6 positive (n = 33) (n = 31) p value Sex 0.3474^(d)Male 32 28 Female 1 3 HIV risk factors 0.6673^(d) Homosexual 31 28Others 2 3 Number of KS lesions 0.1321^(d) None 12 18 Any 21 13  1–10 11 0.297^(f) 10–50 2 4 >50 18 8 KS severity 0.307^(f) No KS 12 18 T0 5 7T1 16 6 KS treatment 1.0000^(d) None 17 16 Any 16 15 anti-herpesvirusagents 7 11 0.2691^(d) others 15 7 0.0686^(d) Anti-retroviral agents1.0000^(d) None 7 6 Any 26 25 NRTI 26 24 1.0000^(d) NNRTI 2 3 0.6673^(d)PI 9 11 0.5919^(d) Lymphocyte counts CD4⁺ T cells (/μL) 155 ± 181 181 ±190 0.3825^(e) CD8⁺ T cells (/μL) 614 ± 312 703 ± 467 0.7168^(e) CD4/8ratio 0.25 ± 0.38 0.23 ± 0.18 0.2677^(e) HIV RNA (copies/mL)   47329 ±110189^(b)  49261 ± 99280^(c) 0.9471^(e) Lymphadenopathy 7 2 0.1498^(d)Platelet counts (/μL) 195 ± 80  202 ± 63  0.2538^(e) ^(a)Allparticipants were enrolled in clinical studies at National CancerInstitute, Bethesda, MD. ^(b)n = 18 ^(c)n = 17 ^(d)Statisticaldifference in the subgroup was determined by Fisher's exact test.^(e)Statistical difference was determined by Mann-Whitney U test.^(f)v-value by Cramer's test in the subgroup is shown. NRTI nucleosidereverse transcriptase inhibitors, NNRTI non-nucleoside reversetranscriptase inhibitors, PI protease inhibitor

No direct association was noted between vIL-6 levels and KS severity(p=0.1321, Fisher's exact test), HIV RNA load (p=0.9471, Mann-Whitney Utest), CD4 cell counts (p=0.3825, Mann-Whitney U test), andlymphadenopathy p=0.1498, Fischer's exact test). These results suggestthat in HIV-infected individuals, circulating vIL-6 is not a criticalfactor in the progression to KS.

KS, a rare malignancy in the general population, is the leading neoplasmin AIDS occurring in approximately 20% of homosexual and bisexualpatients. DNA-based and serology-based studies have demonstrated aconsistent association of KSHV with both AIDS-related and AIDS-unrelatedforms of KS (Antman et al., N Engl J Med 342:1027–38, 2000). Thisinformation and other evidence suggest that KSHV is an essentialetiologic agent to the development of KS. Earlier studies have shownthat the KSHV-encoded cytokine vIL-6, an early lytic viral gene, is onlyrarely expressed in KS lesions. In the present study, we detected vIL-6in 16 of 35 (46%) of serum samples from North American patients withAIDS-associated KS. As expected, we found vIL-6 to be rarely detectablein North American blood donors. However, nearly 57% of patients withAIDS without KS, PEL or MCD had detectable vIL-6 in the circulation.Thus, AIDS patients in this study frequently displayed vIL-6 in thecirculation regardless of the presence or severity of KS. These resultsare consistent with previous studies showing that KSHV replicates inonly a minority of spindle cells within KS lesions, and further suggestthat KSHV frequently replicates and expresses vIL-6 at some site inKSHV-infected AIDS patients.

Example 12 Producing an Immune Response Against vIL-6

In one embodiment, a method of treating a subject with Kaposi'ssarcoma-associated herpes virus (KSHV) associated disorder is provided,or preventing or inhibiting the development of clinical disease.Alternatively, the method can be used to inhibit the progress of analready existing KSHV disorder. The method includes administering to thesubject a therapeutically effective amount a polypeptide including theC-terminal region of the AB loop and/or helix B of the Kaposi'ssarcoma-associated herpes virus IL-6polypeptide, in order to produce animmune response. The immune response thereby treats or prevents theinfection, or retards or reverses clinical disease. In one embodiment,the polypeptide consists of the C-terminal region of the AB loop andhelix B of the vIL-6 alone. In another embodiment, the C-terminal regionof the AB loop and helix B of the vIL-6 is conjugated with a carrier, oradministered with an ajuvant.

In forming a composition for generating an immune response in a subject,or for vaccinating a subject, C-terminal region of the AB loop and helixB of the vIL-6, or a derivative or variant thereof, is utilized. Analogsinvolving amino acid deletions, amino acid replacements (e.g.,conservative substitutions), or by isostereomer (a modified amino acidthat bears close structural and spatial similarity to the original aminoacid) substitutions, isostereomer additions, and amino acid additionscan be utilized, so long as the sequences elicit an immune responseagainst vIL-6, such as antibodies that specifically bind the vIL-6.

In the formation of a peptide derived from natural sources, a proteinincluding an amino acid sequence described herein (e.g. the vIL-6) issubject to selective proteolysis. Selective proteolysis includessplitting the protein with chemical reagents or enzymes. Alternatively,a peptide that consists essentially of the C-terminal region of the ABloop and/or helix B of the vIL-6 as described herein can be chemicallysynthesized. In one embodiment, the peptide is synthesized in propersynthetic configuration to be recognized by a monoclonal antibodysecreted by the hybridoma v6 m12.1.1, v6 m17.3.2, vm31.2.4 or v6m27.1.2.

The C-terminal region of the AB loop and/or helix B of the vIL-6 canalso be engineered to include other amino acids, such as residues ofvarious moieties, such as additional amino acid segments orpolysaccharides. In addition, an amino acid chain corresponding to anadditional antigen or immunogen can be included. Thus, an immuneresponse to more than one antigen can be induced by immunization.Specific non-limiting examples of antigens or immunogens include, butare not limited to, antigens of an immunodeficiency virus, hepatitis B,measles, influenza, smallpox, polio, or diptheria. These additionalamino acid sequences can be of varying length.

The sequences of amino acids can be interconnected with one another suchas by cross-linking or can be bound together covalently. Alternatively,an immunogenic composition can be an admixture with other proteins thatare known immunogens. In one embodiment, the peptides included in thecomposition are capable of forming neutralizing antibodies to the KSHV.

A carrier may be provided for the C-terminal region of the AB loopand/or helix B of the vIL-6 disclosed herein. However, a carrier may notbe required to induce an immune response to the polypeptide. A “carrier”is a physiologically acceptable mass to which the C-terminal region ofthe AB loop and/or helix B of the vIL-6 is attached and which isexpected to enhance the immune response. In one embodiment, a carrier isa chain of amino acids or other moieties. In another embodiment, acarrier is a dimer, oligomer, or higher molecular weight polymer of asequence of amino acids of the C-terminal region of the AB loop and/orhelix B of the vIL-6. In other words, the polypeptide can be formed fromnaturally available materials or synthetically produced and can then bepolymerized to build a chain of two or more repeating units so that therepeating sequences form both the carrier and the immunogenicpolypeptide. Alternatively, additional amino acids can be added to oneor both ends of the C-terminal region of the AB loop and/or helix B ofthe vIL-6.

Alternative carriers are some substance, animal, vegetable, or mineralin origin, that is physiologically acceptable and functions to presentthe C-terminal region of the AB loop and/or helix B of the vIL-6 to theimmune system. Thus, a wide variety of carriers are acceptable, andinclude materials which are inert, or which have biological activityand/or promote an immune response. For example, an example of a proteincarrier includes, but is not limited to, keyhole lympet protein, andhemocyanin. Polysaccharides can also be used as carriers, and includethose of molecular weight 10,000 to 1,000,000, such as starches,dextran, agarose, ficoll, or it's carboxylmethyl derivative and carboxymethyl cellulose.

Polyamino acids are also contemplated for use as carriers, and thesepolyamino acids include, among others, polylysine, polyalanylpolylysine, polyglutamic acid, polyaspartic acid and poly (C₂–C₁₀) aminoacids.

Organic polymers can be used as carriers, and these polymers include,for example, polymers and copolymers of amines, amides, olefins, vinyls,esters, acetals, polyamides, carbonates and ethers and the like.Generally speaking, the molecular weight of these polymers will varydramatically. The polymers can have from two repeating units up toseveral thousand, e.g., two thousand repeating units. The number ofrepeating units will be consistent with the use of the immunizingcomposition in a host animal. Generally speaking, such polymers willhave a lower molecular weight, say between 10,000 and 100,000 (themolecular weight being determined by ultracentrifugation).

Inorganic polymers can also be employed. These inorganic polymers can beinorganic polymers containing organic moieties. In particular, silicatesand aluminum hydroxide can be used as carriers. In one embodiment, thecarrier is one which is an immunological adjuvant. In such cases, theadjuvant can be muramyl dipeptide or its analogs.

The carrier can also be the residue of a crosslinking agent employed tointerconnect a plurality of synthetic peptide containing chains.Crosslinking agents which have as their functional group an aldehyde(such as glutaraldehyde), carboxyl, amine, amido, imido or azidophenylgroup. In particular, there is contemplated the use of butyraldehyde asa crosslinking agent, a divalent imido ester or a carbodiimide.

Chemical synthesis of peptides is described in the followingpublications: S. B. H. Kent, Biomedical Polymers, eds. Goldberg andNakajima, Academic Press, New York, pp. 213–242, 1980; Mitchell et al.,J. Org. Chem., 43, 2845–2852, 1978; Tam, et al., Tet. Letters,4033–4036, 1979; Mojsov, A. R. Mitchell, and R. B. Merrifield, J. Org.Chem., 45, 555–560, 1980; Tam et al., Tet. Letters, 2851–2854, 1981; andKent et al., Proceedings of the IV International Symposium on Methods ofProtein Sequence Analysis, (Brookhaven Press, Brookhaven, N.Y., 1981.

In one embodiment, the method is provided for administering to a subjecta therapeutically effective amount of a nucleic acid encoding C-terminalregion of the AB loop and/or helix B of the vIL-6, thereby treatingproducing an immune response against the vIL-6. Specific, non-limitingexamples of an immune response are a B cell or a T cell response.

For administration of nucleic acids molecules, various viral vectors canbe utilized. These vectors include adenovirus, herpes virus, vaccinia,or an RNA virus such as a retrovirus. In one embodiment, the retroviralvector is a derivative of a murine or avian retrovirus. Examples ofretroviral vectors in which a single foreign gene can be insertedinclude, but are not limited to: Moloney murine leukemia virus (MoMuLV),Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus(MuMTV), and Rous Sarcoma Virus (RSV). When the subject is a human, avector such as the gibbon ape leukemia virus (GaLV) can be utilized. Anumber of additional retroviral vectors can incorporate multiple genes.All of these vectors can transfer or incorporate a gene for a selectablemarker so that transduced cells can be identified and generated. Byinserting a nucleic acid sequence encoding the C-terminal region of theAB loop and/or helix B of the vIL-6 into the viral vector, along withanother gene which encodes the ligand for a receptor on a specifictarget cell, for example, the vector is now target specific. Retroviralvectors can be made target specific by attaching, for example, a sugar,a glycolipid, or a protein. Preferred targeting is accomplished by usingan antibody to target the retroviral vector. Those of skill in the artwill know of, or can readily ascertain without undue experimentation,specific polynucleotide sequences which can be inserted into theretroviral genome or attached to a viral envelope to allow targetspecific delivery of the retroviral vector containing the polynucleotideencoding the C-terminal region of the AB loop and/or helix B of thevIL-6.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include, but are not limited to Q2,PA317, and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for the therapeutic polynucleotides isa colloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. One colloidal system of this invention is aliposome. Liposomes are artificial membrane vesicles that are useful asdelivery vehicles in vitro and in vivo. It has been shown that largeuni-lamellar vesicles (LV), which range in size from 0.2–4.0 μm canencapsulate a substantial percentage of an aqueous buffer containinglarge macromolecules. RNA, DNA and intact virions can be encapsulatedwithin the aqueous interior and be delivered to cells in a biologicallyactive form (Fraley et al., 1981, Trends Biochem. Sci. 6:77, 1981). Inaddition to mammalian cells, liposomes have been used for delivery ofpolynucleotides in plant, yeast and bacterial cells. In order for aliposome to be an efficient gene transfer vehicle, the followingcharacteristics should be present: (1) encapsulation of the genes ofinterest at high efficiency while not compromising their biologicalactivity; (2) preferential and substantial binding to a target cell incomparison to non-target cells; (3) delivery of the aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4)accurate and effective expression of genetic information (Mannino etal., Biotechniques 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidyl-glycerols, where the lipid moiety contains from 14–18carbon atoms, particularly from 16–18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

The present disclosure involves administering to a subject atherapeutically effective dose of a pharmaceutical compositioncontaining a nucleic acid encoding a C. the C-terminal region of the ABloop and/or helix B of the vIL-6 and a pharmaceutically acceptablecarrier. Administering the pharmaceutical composition of the presentinvention may be accomplished by any means known to the skilled artisan.By subject is meant any mammal, including a human.

Example 13 Pharmaceutical Compositions and Modes of Administration

Various delivery systems for administering the specific binding agentsfor vIL-6 are known. Similarly, various delivery systems foradministering or C-terminal region of the AB loop and/or helix B of thevIL-6 polypeptide are known. These delivery systems include e.g.,encapsulation in liposomes, microparticles, microcapsules, expression byrecombinant cells, receptor-mediated endocytosis (see, e.g., Wu and Wu,J. Biol. Chem. 62:4429–32, 1987), construction of a therapeutic nucleicacid as part of a retroviral or other vector, etc. Methods ofintroduction include but are not limited to intradermal, intramuscular,intraperitoneal, intrapleural, intravenous, subcutaneous, intranasal,and oral routes. The compounds may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, the pharmaceutical compositions may be introduced into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment, for example, by local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, through a catheter, by a suppository or an implant, suchas a porous, non-porous, or gelatinous material, including membranes,such as silastic membranes, or fibers. In one embodiment, administrationcan be by direct injection at the site (or former site) of a malignanttumor or neoplastic or pre-neoplastic tissue, such as a Kaposi's sarcomalesion. In a specific embodiment, administration is performed directlyinto a vIL-6-expressing cell.

The use of liposomes as a delivery vehicle is one delivery method ofinterest. The liposomes fuse with the target site and deliver thecontents of the lumen intracellularly. The liposomes are maintained incontact with the target cells for a sufficient time for fusion to occur,using various means to maintain contact, such as isolation and bindingagents. Liposomes may be prepared with purified proteins or peptidesthat mediate fusion of membranes, such as Sendai virus or influenzavirus. The lipids may be any useful combination of known liposomeforming lipids, including cationic lipids, such as phosphatidylcholine.Other potential lipids include neutral lipids, such as cholesterol,phosphatidyl serine, phosphatidyl glycerol, and the like. For preparingthe liposomes, the procedure described by Kato et al. (J. Biol. Chem.266:3361, 1991) may be used.

In an embodiment where the therapeutic molecule is an antibody,specifically a monoclonal antibody (mAb), combined with adifferentiation-inducing agent, administration may be achieved by directinjection, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents.

The present invention also provides pharmaceutical compositions whichinclude a therapeutically effective amount of the inducing agent with anmAb that neutralizes a biological function of vIL-6, and apharmaceutically acceptable carrier or excipient. Such carriers include,but are not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The carrier and compositioncan be sterile, and the formulation suits the mode of administration.The composition can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. The composition can be a liquidsolution, suspension, emulsion, tablet, pill, capsule, sustained releaseformulation, or powder. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulations can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate.

In a particular embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule, indicating the quantity of active agent.Where the composition is to be administered by infusion, it can bedispensed with an infusion bottle containing sterile pharmaceuticalgrade water or saline.

The compositions can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with free carboxyl groupssuch as those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, and procaine. The amount of the inducing agent and disruptingagent that will be effective in the treatment of a particular disorderor condition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. In addition, in vitroassays may optionally be employed to help identify optimal dosageranges. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test systems.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% of theactive ingredients. The invention also provides a pharmaceutical pack orkit comprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions. Optionally associatedwith such container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

The pharmaceutical compositions or methods of treatment may beadministered in combination with other therapeutic treatments, such asother anti-neoplastic anti-inflammatory, anti-viral or other therapies.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that the illustratedembodiment is only a preferred example of the invention and should notbe taken as a limitation on the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. A method for treating a subject infected with a Kaposi'ssarcoma-associated herpesvirus (KSHV), comprising administering atherapeutically effective amount of a monoclonal antibody thatspecifically binds Kaposi's sarcoma-associated herpesvirus (KSHV)interleukin-6 (vIL-6), wherein the monoclonal antibody is produced by ahybridoma deposited as American Type Culture Collection (ATCC) DepositNo. PTA-2217 PTA-2218, PTA-2219, or PTA-2220 or a humanized form of themonoclonal antibody, in a pharmaceutically acceptable carrier.
 2. Themethod of claim 1, wherein the subject has primary effusion lymphoma,Castleman's disease, or Kaposi's sarcoma.
 3. The method of claim 1,wherein the administration is by subcutaneous, intra-vascular,intra-peritoneal, intra-pleural, or intra-muscular injection.
 4. Themethod of claim 1, wherein the subject is infected with humanimmunodeficiency virus (HIV).
 5. The method of claim 1, wherein themonoclonal antibody is a humanized form of the hybridoma deposited asAmerican Type Culture Collection (ATCC) Deposit No. PTA-2217, PTA-2218,PTA-2219, or PTA-2220.
 6. The method of claim 1, wherein the monoclonalantibody is produced by a hybridoma deposited as America Type CultureCollection (ATTC) Deposit No. PTA-2217, PTA-2218, PTA-2219, or PTA-2220.7. The method of claim 1, wherein the monoclonal antibody is produced bya hybridoma deposited as American Type Culture Collection (ATCC) DepositNo. PTA-2217.
 8. The method of claim 1, wherein the monoclonal antibodyis produced by a hybridoma deposited as American Type Culture Collection(ATCC) Deposit No. PTA-2218.
 9. The method of claim 1, wherein themonoclonal antibody is produced by a hybridoma deposited as AmericanType Culture Collection (ATCC) Deposit No. PTA-2219.
 10. The method ofclaim 1, wherein the monoclonal antibody is a humanized form of themonoclonal antibody produced by a hybridoma deposited as American TypeCulture Collection (ATCC) Deposit No. PTA-2217.
 11. The method of claim1, wherein the monoclonal antibody is a humanized form of the monoclonalantibody produced by a hybridoma deposited as American Type CultureCollection (ATCC) Deposit No. PTA-2218.
 12. The method of claim 1,wherein the monoclonal antibody is a humanized form of the monoclonalantibody produced by a hybridoma deposited as American Type CultureCollection (ATCC) Deposit No. PTA-2219.